CBI analogs of CC-1065 and the duocarmycins

ABSTRACT

Analogs of the antitumor antibiotics CC-1065 and the duocarmycins incorporate the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) alkylation subunit. The CBI-based analogs have potent cytotoxic activity and are useful as efficacious antitumor compounds. A direct relationship between functional stability and in vitro cytotoxic potency is disclosed. The CBI-based analogs are easily synthesized and are 4× more stable and 4× more potent than the corresponding analogs containing the authentic CPI alkylation subunit of CC-1065 and comparable in potency to agents containing the authentic alkylation subunit of duocarmycin SA. Similarly, the CBI-based agents alkylate DNA with an unaltered sequence selectivity at an enhanced rate and with a greater efficiency than the corresponding CPI analog and were comparable to the corresponding analog incorporating the duocarmycin SA alkylation subunit. Systematic and extensive modifications and simplifications in the DNA binding subunits attached to CBI are also described.

FIELD OF INVENTION

The invention relates to antitumor antibiotics. More particularly, theinvention relates to analogs of CC-1065 and the duocarmycins havingantitumor antibiotic activity.

BACKGROUND

(+)-CC-1065 (1) and the duocarmycins 2-3 represent the initial membersof a class of exceptionally potent antitumor antibiotics. Members ofthis class of antitumor antibiotic derive their biological effectsthrough the reversible, stereoelectronically-controlled sequenceselective alkylation of duplex DNA. (H. Sugiyama, et al., TetrahedronLett. 1990, 31, 7197; C. H. Lin, et al., J. Am. Chem. Soc. 1992, 114,10658; H. Sugiyama, et al., Tetrahedron Lett. 1993, 34, 2179; K.Yamamoto, et al., Biochemistry 1993, 32, 1059; A. Asai, et al., J. Am.Chem. Soc. 1994, 116, 4171; and D. L. Boger, et al., Tetrahedron 1991,47, 2661.) (+)-CC-1065 (1) was first disclosed in 1981 by L. J. Hanka,et al. (J. Am. Chem. Soc. 1981, 103, 7629.) The duocarmycins 2-3 werefirst disclosed in 1988 and 1990. (Takahashi, et al. J. Antibiot. 1988,41, 1915; T. Yasuzawa, et al., Chem. Pharm. Bull. 1988, 36, 3728; M.Ichimura, et al., J. Antibiot. 1988, 41, 1285; M. Ichimura, et al., J.Antibiot. 1990, 43, 1037; M. H. Ichimura, et al., J. Antibiot. 1991, 44,1045; K. Ohba, et al., J. Antibiot. 1988, 41, 1515; and S. Ishii, J.Antibiot. 1989, 42, 1713.)

Subsequent to their disclosure, extensive efforts have been devoted toestablish their duplex DNA alkylation selectivity and its structuralorigin. (D. L. Boger, Acc. Chem. Res. 1995, 28, 20; D. L. Boger, Proc.Natl. Sci. U.S.A. in press; D. L. Boger, Chemtracts: Org. Chem. 1991, 4,329; D. L. Boger, In Proceed R. A. Welch Found. Conf. on Chem. Res.,XXXV. Chem. at the Frontiers of Medicine 1991, 35, 137; D. L. Boger, InAdvances in Heterocyclic Natural Products Synthesis, Vol. 2, Pearson, W.H. Ed.; JAI Press: Greenwich, Conn., 1992, 1-188; D. L. Boger, PureAppl. Chem. 1993, 65, 1123; D. L. Boger, Pure Appl. Chem. 1994, 66, 837;R. S. Coleman, In Studies in Nat. Prod. Chem., Vol 3, Rahman, A.-u.-,Ed.; Elsevier: Amsterdam, 1989, 301; and D. L. Boger, In Heterocycles inBioorganic Chemistry; J. Bergman, H. C. van der Plas, and M. Simonyl,Eds; Royal Society of Chemistry: Cambridge, 1991, 103.) Progress hasalso been made with respect to characterizing the link between DNAalkylation and the ensuing biological properties. (D. L. Boger, et al.,Bioorg. Med. Chem. Lett. 1994, 4, 631.) Extensive efforts have also beendevoted to define the fundamental principles underlying therelationships between structure, chemical reactivity, and biologicalproperties. (W. Wierenga, et al., Adv. Enzyme Regul. 1986, 25, 141; M.A. Warpehoski, et al., J. Med. Chem. 1988, 31, 590; D. L. Boger, et al.,J. Am. Chem. Soc. 1993, 115, 9025; D. L. Boger, et al., J. Am. Chem.Soc. 1992, 114, 10056; H. Muratake, et al., Tetrahedron Lett. 1994, 35,2573; Y. Fukuda, et al., Tetrahedron 1994, 50, 2793; Y. Fukuda, et al.,Tetrahedron 1994, 50, 2809; Y. Fukuda, et al., Bioorg. Med. Chem. Lett.1992, 2, 755; Y. Fukuda, et al., Tetrahedron Lett. 1990, 31, 6699; W.Wierenga, J. Am. Chem. Soc. 1981, 103, 5621; P. Magnus, et al., J. Am.Chem. Soc. 1987, 109, 2706; G. A. Kraus, et al., J. Org. Chem. 1985, 50,283; D. L. Boger, et al., J. Am. Chem. Soc. 1988, 110, 1321, 4796; R. E.Bolton, et al., J. Chem. Soc., Perkin Trans. 1 1988, 2491; R. J.Sundberg, et al., J. Org. Chem. 1988, 53, 5097; R. J. Sundberg, et al.,J. Org. Chem. 1991, 56, 3048; V. P. Martin, Helv. Chim. Acta 1989, 72,1554; M. Toyota, et al., J. Chem. Soc., Perkin Trans. 1 1992, 547; andL. F. Tietze, et al., J. Org. Chem. 1994, 59, 192.) The relationshipsbetween structure, chemical reactivity, and biological properties ofCI-based analogs have also been characterized. (D. L. Boger, et al.,Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1431; D. L. Boger, et al., J.Am. Chem. Soc. 1991, 113, 3980; D. L. Boger, et al., J. Org. Chem. 1989,54, 1238; D. L. Boger, et al., J. Am. Chem. Soc. 1990, 112, 5230; K. J.Drost, et al., J. Org. Chem. 1989, 54, 5985; J. H. Tidwell, et al., J.Org. Chem. 1992, 57, 6380; J. Sundberg, et al., Tetrahedron Lett. 1986,27, 2687; Y. Wang, et al., Heterocycles 1993, 36, 1399; Y. Wang, et al.,J. Med. Chem. 1993, 36, 4172; L. F. Tietze, et al., Chem. Ber. 1993,126, 2733; and T. Sakamoto, et al., J. Chem. Soc., Perkin Trans. 1 1993,1941.) The relationships between structure, chemical reactivity, andbiological properties of C₂BI-based analogs have also beencharacterized. (D. L. Boger, et al., J. Am. Chem. Soc. 1992, 114, 9318;and D. L. Boger, et al., Bioorg. Med. Chem. 1993, 1, 27.) Therelationships between structure, chemical reactivity, and biologicalproperties of CBQ-based analogs have also been characterized. (D. L.Boger, et al., J. Am. Chem. Soc. 1994, 116, 6461; and D. L. Boger, etal., J. Am. Chem. Soc. 1994, 116, 11335.) F. Mohamadi et al. havecharacterized the relationships between structure, chemical reactivity,and biological properties of CFI-based analogs (J. Med. Chem. 1994, 37,232.) A p-quinonemethide analog was characterized by D. L. Boger, et al.(J. Org. Chem. 1994, 59, 4943.)

Concurrent with the above structure/function studies, substantialefforts have been devoted to developing potential clinical candidatesbased on the natural product structures having enhanced in vivoefficacy. Compounds 4-8 are analogs of the natural product structureshaving enhanced in vivo efficacy with clinical potential. (D. L. Boger,et al., J. Org. Chem. 1984, 49, 2240; M. A. Warephoski, M. A.Tetrahedron Lett. 1986, 27, 4103; Li, L. H.; Invest. New Drugs 1991, 9,137; B. K. Bhuyan, et al., Cancer Res. 1992, 52, 5687; B. K. Bhuyan, etal., Cancer Res. 1993, 53, 1354; L. H. Li, et al., Cancer Res. 1992, 52,4904; M. A. Mitchell, et al., J. Am. Chem. Soc. 1991, 113, 8994. Lee,C.-S.; Gibson, N. W. Cancer Res. 1991, 51, 6586. Lee, C.-S.; Gibson, N.W. Biochemistry 1993, 32, 9108; Wierenga, W. Drugs Fut. 1991, 16, 741;K. Gomi, et al., Jpn. J. Cancer Res. 1992, 83, 113. Okamoto, A.; Okabe,M.; Gomi, K. Jpn. J. Cancer Res. 1993, 84, 93; E. Kobayashi, et al.,Cancer Res. 1994, 54, 2404; and H. Ogasawara, Jpn. J. Cancer Res. 1994,85, 418.) A Phase I clinical trial one one drug candidate in this classis described by G. F. Fleming, et al., (J. Natl. Cancer Inst. 1994, 86,368.) Efforts have also focused on the development of analogs havingdecreased delayed toxicity as compared to the natural form of(+)-CC-1065. (J. P. McGovren, et al., Cancer Res. 1993, 53, 5690.)Importantly, this unusual property has not been observed withent-(−)-CC-1065, although it is equally cytotoxic, and is not observedwith the naturally-derived duocarmycins as well as simplified analogs ofthe natural products.

The first preparation and examination of agents containing the1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) alkylationsubunit were described in connection with efforts to evaluate CC-1065and duocarmycin analogs bearing deep-seated structural alterations inthe alkylation subunit. (D. L. Boger, et al., J. Am. Chem. Soc. 1989,111, 6461; and D. L. Boger, et al., J. Org. Chem. 1990, 55, 5823.) Theseagents were employed as tools to identify the structural features ofcompounds 1-3 associated with their sequence selective alkylation ofduplex DNA and to define the fundamental relationships betweenstructure, chemical or functional reactivity and biological properties.

Prior to the present invention, it had been assumed that the uniquealkylating activity of the naturally occurring CPI subunit of CC-1065would be degraded if this portion of the molecule were structurallyaltered. (L. H. Hurley, et al., Science 1984, 226, 843; V. L. Reynolds,et al., Biochemistry 1985, 24, 6228. L. H. Hurley, et al., Biochemistry1988, 27, 3886; L. H. Hurley, et al., J. Am. Chem. Soc. 1990, 112, 4633;M. A. Warpehoski, et al., J. Biochemistry 1992, 31, 2502; D. L. Boger,et al., Bioorg. Med. Chem. 1994, 2, 115; D. L. Boger, et al., J. Am.Chem. Soc. 1990, 112, 4623; M. A. Warpehoski, et al., In Advances in DNASequence Specific Agents; Hurley, L. H., Ed.; JAI Press: Greenwich,Conn., 1992, Vol 1, 217; M. A. Warpehoski, Drugs Fut. 1991, 16, 131; M.A. Warpehoski, et al., in Molecular Basis of Specificity in NucleicAcid-Drug Interactions; B. Pullman and J. Jortner, Eds.; Kluwer:Netherlands; 1990, 531; M. A. Warpehoski, et al., Chem. Res. Toxicol.1988, 1, 315; Hurley, L. H.;. In Molecular Aspects of AnticancerDrug-DNA Interactions; Neidle, S., Waring, M., Eds.; CRC Press: AnnArbor, MI 1993, Vol 1, 89; and L. H. Hurley, et al., Acc. Chem. Res.1986, 19, 230.) The above assumption is disclosed herein to beinaccurate. Futhermore, the natural enantiomers of the CBI-based analogsof (+)-CC-1065, have been shown to be approximately four times morestable chemically and approximately four times more potent biologicallyas compared to the corresponding agents incorporating the natural CPIalkylation subunit of CC-1065. (D. L. Boger, et al., Tetrahedron Lett.1990, 31, 793; D. L. Boger, et al., J. Org. Chem. 1992, 57, 2873; and D.L. Boger, et al., J. Org. Chem. 1995, 60, 0000.) The CBI analogs arealso considerably more synthetically accessible as compared to thenaturally occuring CPI compounds. (+)-CBI-indole₂ (27) exhibitscytotoxic potency comparable to that of the (+)-CC-1065 and greater (4×)than that of the potential clinical candidate (+)-CPI-indole₂ (4,U71,184) introduced by Upjohn. (+)-CBI-indole₂ (27) also exhibits potentand efficacious in vivo antitumor activity. (D. L. Boger, et al.,Bioorg. Med. Chem. Lett. 1991, 1, 115.) (+)-CBI-indole₂ (27) was thefirst efficacious antitumor activity by a CC-1065 analog possessing astructurally altered and simplified DNA alkylation subunit. Moreover,the agent further lacked the delayed fatal toxicity characteristic of(+)-CC-1065.

The natural enantiomers of the CBI-based analogs have been shown toalkylate DNA with an unaltered sequence selectivity as compared to thecorresponding CPI analog. (D. L. Boger, et al., J. Am. Chem. Soc. 1994,116, 7996; and P. A. Aristoff, et al., J. Med. Chem. 1993, 36, 1956.)Furthermore, the DNA alkylation of CBI-based analogs occurs at anenhanced rate as compared to the corresponding CPI analogs (D. L. Boger,et al., J. Am. Chem. Soc. 1991, 113, 2779) and with a greater efficiencythan the corresponding CPI analog. (D. L. Boger, et al., J. Am. Chem.Soc. 1992, 114, 5487)

Refined models of the DNA alkylation reactions of the duocarmycins havebeen developed which accomodate the reversed and offset AT-rich adenineN3 DNA alkylation selectivity of the enantiomeric agents and theirstructural analogs. (D. L. Boger, et al., J. Org. Chem. 1990, 55, 4499;D. L. Boger, et al., J. Am. Chem. Soc. 1990, 112, 8961; D. L. Boger, etal., J. Am. Chem. Soc. 1991, 113, 6645; D. L. Boger, et al., J. Am.Chem. Soc. 1993, 115, 9872; D. L. Boger, et al., Bioorg. Med. Chem.Lett. 1992, 2, 759; and D. L. Boger, et al., J. Am. Chem. Soc. 1994,116, 1635.) A similar refined model of the DNA alkylation reactions ofCC-1065 have been developed which also accomodate the reversed andoffset AT-rich adenine N3 DNA alkylation selectivity of the enantiomericagents and their structural analogs. (D. L. Boger, et al., Bioorg. Med.Chem. 1994, 2, 115; and D. L. Boger, et al., J. Am. Chem. Soc. 1990,112, 4623.) These models teach that the diastereomeric adducts derivedfrom the unnatural enantiomers suffer a significant destabilizing stericinteraction between the CPI C7 center (CH₃) or the CBI C8 center withthe base adjacent to the alkylated adenine which is not present with thenatural enantiomer adducts. Moreover, the distinguishing features of thenatural and unnatural enantiomers diminish or disappear as the inherentsteric bulk surrounding this center is reduced or removed. Because ofthe unnatural enantiomer sensitivity to destabilizing stericinteractions surrounding the CPI C7 or CBI C8 center, the unnaturalenantiomers of the CBI-based analogs are particularly more effectivethan the corresponding CPI analog displaying an even more enhancedrelative rate and efficiency of DNA alkylation.

SUMMARY

An extensive study of analogs of the potent antitumor antibioticsCC-1065 and the duocarmycins which incorporate the1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) alkylationsubunit are detailed. In contrast to early speculation, deep-seatedmodifications in the CC-1065 and duocarmycin alkylation subunits arewell tolerated and the CBI-based analogs proved to be potent cytotoxicagents and efficacious antitumor compounds. Full details of studiesdefining a direct relationship between functional stability and in vitrocytotoxic potency are described. As such, the readily accessibleCBI-based analogs were found to be 4× more stable and 4× more potentthan the corresponding analogs containing the authentic CPI alkylationsubunit of CC-1065 and comparable in potency to agents containing theauthentic alkylation subunit of duocarmycin SA. Similarly, the CBI-basedagents alkylate DNA with an unaltered sequence selectivity at anenhanced rate and with a greater efficiency than the corresponding CPIanalog and were comparable to the corresponding analog incorporating theduocarmycin SA alkylation subunit. Systematic and extensivemodifications and simplifications in the DNA binding subunits attachedto CBI were explored with the comparisons of both enantiomers of 1-3with both enantiomers of 18-24, 25-29, 57-61, 62-65, 66-68, 72-73 and78-79.

CPI and CBI Structures

Simple Derivatives of CBI. Role of the N² Substituent and Validation ofa Direct Relationship Between Functional Stability and In VitroCytotoxic Potency. Substantial quantities of optically activenatural-(1S)- and ent-(1R)-15 were prepared through use of our originalsynthesis of CBI and its precursors, as referenced above with two recentmodifications. (D. L. Boger, et al., J. Org. Chem. 1992, 57, 2873; andD. L. Boger, et al., J. Org. Chem. 1995, 60, 0000.) The most efficientapproach now proceeds in 9 steps and in 38% overall yield fromcommercially available 1,3-dihydroxynapthalene based on a key 5-exo-trigaryl radical-alkene cyclization for the direct preparation ofN-BOC-5-benzyloxy-1-hydroxymethyl-1,2-dihydro-3H-benz[e]indole.Moreover, the initial resolution we described based on thechromatographic separation of the diastereomeric (R)-O-acetyl madelateesters of the primary alcohol precursor to 15 which has been adopted byothers has been since improved in our efforts. The more advancedsynthetic intermediate 15, and in fact the penultimate intermediate tothe CBI-based analogs, may be directly and more efficiently resolved(α=1.28) on an analytical or preparative Daicel Chiralcel OD columnwithout recourse to diastereomeric derivatization. For our purposes, 20mg of 15 could be separated in a single injection on a semipreparative10 μm, 2×25 cm OD HPLC column (5% i-PrOH-hexane, 8 mL/min) with a90-100% recovery of the total sample. Conversion of natural (1S)- andent-(1R)-15 to (+)- and ent-(−)-N-BOC-CBI (9), and (+)- and ent-(−)-CBI(17) have been detailed in our initial studies, and provided ourcomparison standards for the studies detailed below (FIG. 1).

Initial studies conducted with simple derivatives of the (+)-CC-1065alkylation subunit (CPI) led to the proposal that there exists a directrelationship between an agent's reactivity and in vitro cytotoxicpotency (L1210, IC₅₀) and established the expectation that thebiological potency may be enhanced as their electrophilic reactivity isincreased. In our complementary series of studies conducted with agentscontaining deep-seated modifications in the alkylation subunit including9-14, the reverse relationship has been observed and the agentspossessing the greatest chemical solvolysis stability exhibited the mostpotent in vitro cytotoxic activity. Moreover, a direct relationshipbetween solvolytic stability and biological potency has been observedand proved to be general with both simple and advanced analogs of thenatural products.

As a consequence of these studies, we became interested in the inherentrole of the CC-1065 and duocarmycin N² substituent. Consequently, thesimple derivatives 21-24 of (+)-CBI were prepared for examination and,by virtue of their structural similarities, were expected to moreaccurately reflect a potential relationship between functionalreactivity and-biological potency than the preceding studies. Treatmentof crude, freshly prepared 16 with methyl isocyanate (2 equiv, 3 equivNaHCO₃, THF, 0-25° C., 1 h, 83%) provided 18 and attempts to conductthis reaction in more polar solvents including DMF or in the presence ofa stronger base (i.e. Et₃N) which promotes competitive closure of 16 toCBI (17) led to lower conversions. Spirocyclization of 18 to 21 waseffected by treatment with DBU (2 equiv, DMF, 4° C., 48 h, 90%) and theuse of shorter reaction periods (24 h, 55%) or less polar solvents (THF,18 h, 35%) provided lower conversions. Treatment of the freshlygenerated crude indoline hydrochloride salt 16 with ClCO₂CH₃ (2 equiv, 3equiv NAHCO₃, THF, 0-25° C., 1.5 h) provided 19 (100%) in quantitativeconversion. Spirocyclization of 19 to provide 22 was effected bytreatment with DBU (2 equiv, THF, 0° C., 48 h and 25° C., 10 h, 93%) andthe rate of ring closure of 19 to 22 only became significant at 25° C.under these conditions. Even treatment of 19 with K₂CO₃ (1.5 equiv, THF,25° C., 5 d, 51%) provided 22 albeit with this latter reaction requiringa long reaction period. Similarly, treatment of crude 16 with ClCOCH₂CH₃(2 equiv, 3 equiv NaHCO₃, THF, 0-25° C., 5 h or 0° C., 1H) cleanlyprovided 20 (94-98%). Spirocyclization of 20 to cleanly provide 23 waseffected by simply dissolving 20 in a 1:1 mixture of 5% aqueousNaHCO₃-THF (25° C., 5-10 h, 97%) and stirring the resulting two-phasereaction mixture at room temperature. Given the ease of hydrolysis ofN-acyl-CBI derivatives upon exposure to aqueous base, it is of specialnote that this set of reaction conditions worked so well for 23. Lowerconversions to 23 were observed upon treatment of 20 with DBU (2 equiv,THF, 0-25° C., 18 h) and, although this was not examined in detail, canbe attributed to a slow cyclization under the reaction conditionsresulting in significant amounts of recovered, unreacted 20.Surprisingly, the most challenging of the derivatives to prepare was 24.Attempts to couple freshly generated 16 with ClSO₂CH₂CH₃ under a widerange of reaction conditions deliberately generating or avoiding sulfeneformation suffered from competitive or preferential O-sulfonylation orcompetitive closure to 17. Although this approach could be used togenerate 24, the most productive preparation was accomplished simply byreaction of the sodium salt of CBI (17, 2.5 equiv NaH, THF, 0° C., 10min) with ClSO₂CH₂CH₃ (7 equiv, 3 equiv Et₃N, 25° C., 3 h, 45%) toprovide 24 directly.

The acid-catalyzed solvolysis of 21-24 conducted at pH 3 (CH₃OH—H₂O)were followed spectrophotometrically by UV with the disappearance of thecharacteristic long-wavelength absorption band of the CBI chromophoreand with the appearance of a short-wavelength absorption bandattributable to the seco-N-BOC-CBI derivative, FIGS. 19A-19B. Theresults of these studies along with the cytotoxic activities of 21-24are summarized in FIGS. 20A-20D. The cytotoxic activity of the full setof agents examined and the comparisons with the related CPI-based agentsare summarized in FIG. 7.

The comparisons of 21-24 revealed a direct, linear relationship betweenthe cytotoxic potency (L1210, log 1/IC₅₀) and the solvolytic stability(−log k_(solv), pH 3) of the agents (FIGS. 20A-20D). Thus, similar tothe trend observed with 9-14, the solvolytically more stable derivativesof CBI proved to be the most potent. Similarly, a linear relationshipwas found between the electron-withdrawing properties of the N²substituents (Hammett σ_(p) constant) and the solvolysis reactivity(−log k_(solv), pH 3) of the agents with the strongestelectron-withdrawing substituents providing the most stable agents(FIGS. 20A-20D). This latter relationship reflects the influence of theN² substituent on the ease of C4 carbonyl protonation required forcatalysis of solvolysis and cyclopropyl ring cleavage with the strongerelectron-withdrawing N² substituents exhibiting slower solvolysis rates.Less obvious but more fundamental, the observations were found to followa predictable linear relationship between the cytotoxic potency (L1210,log 1/IC₅₀) and the electron-withdrawing properties of the N²substituent (Hammett σ_(p)) with the strongest electron-withdrawingsubstituents providing the biologically most potent agents (FIGS.20A-20D).

These fundamental correlations between the electron-withdrawingproperties of the N² substituent, the functional reactivity of theagents, and their biological potency should prove useful in thepredictable design of new analogs. In fact, it is this fundamentalvalidation of the direct relationship between functional stability andbiological potency that suggests that the CBI-based analogs, which are4× more stable than the corresponding CPI-based analogs, offerrationally-based advantages that may be expected to be even furtherenhanced by the inherent selectivity that is intrinsic in the diminishedreactivity. For agents in this class which possess sufficient reactivityto effectively alkylate duplex DNA, the chemically more stable agentsmay be expected to constitute the biologically more potent agents.Presumably, this may be attributed to the more effective delivery of themore stable agents to their intracellular target, and the solvolysisrates may be taken to represent a general measure of the relativefunctional reactivity. Notably, the consumption of the agent in route toits intracellular target need not be simply nonproductive solvolysis butcompetitive alkylation of nonproductive extra- and intracellular sitesas well including the potential of nonproductive sites within duplexDNA. Since the chemically more stable agents provide thermodynamicallyless stable and more readily reversed addition products, theobservations may also represent a more effective thermodynamicpartitioning of the agents to their productive intracellular target orsite(s).

Consistent with prior observations, the corresponding seco agents 15 and18-20 which serve as the immediate synthetic precursors to 9 and 21-23exhibited a cytotoxic potency indistinguishable from that of thecorresponding agent incorporating the preformed cyclopropane ring. Sincesimple C4 phenol O-alkyl (CH₃, CH₂Ph) and O-acyl derivatives of 15exhibit substantially diminished cytotoxic potency (10-100×), thisequivalency of the seco precursors 15 and 18-20 with 9 and 21-23 mostlikely may be attributed to their facile closure to the biologicallyrelevant and more potent cyclopropane containing agents. Notably, suchobservations have been instrumental in the successful development ofprodrug strategies for the advanced analogs of the natural productsincluding 6-8.

Although we have described an extensive account of the DNA alkylationproperties of (+)- and ent-(−)-N-BOC-CBI (9) and their comparison withthose of (+)- and ent-(−)-N-BOC-CPI (11) the properties of 21-24 andtheir relationship to the biological evaluations are worth summarizing.The agents 21-24 behaved in a manner comparable to 9. The natural andunnatural enantiomers of 21-24 were substantially less efficient (ca.10⁴×), less selective (selectivity=5′-AA>5′-TA) with 40-45% of alladenines alkylated over a 10-fold agent concentration range, andexhibited an altered DNA alkylation profile than (+)- or ent-(−)-1-3.Moreover, the natural enantiomers of 21-24, like (+)- vs ent-(−)-9,proved to be approximately 5-10× more efficient than the unnaturalenantiomers at alkylating DNA, but were found to exhibit the sameselectivity and alkylate the same sites. This alkylation selectivity of21-24, like that of 9, was identical to that of (+)- orent-(−)-N-BOC-CPI. However, both the natural enantiomers (5×) andespecially the unnatural enantiomers (10-100×) of the CBI-based agentswere more effective at alkylating DNA than the corresponding CPI-basedagent consistent with models that we have discussed in detail.Importantly, the less reactive CBI-based agents were found to alkylateDNA at a faster rate, with a greater efficiency, and with a slightlygreater selectivity among the available sites than the correspondingCPI-based agent. This may be interpreted in terms of agents stericaccessibility to the adenine N3 alkylation site where the C7 methylgroup of the CPI alkylation subunit sterically decelerates the rate ofDNA alkylation to the extent that the less reactive, but moreaccessible, CBI subunit alkylates DNA at a more rapid rate. Since theunnatural enantiomers are even more sensitive to destabilizing stericinteractions at the CPI C7 or CBI C8 position, the unnatural enantiomersof the CBI-based agents are particularly more effective than theCPI-based agents.

Advanced Analogs of CC-1065 and the Duocarmycins: Simplification of theDNA Binding Subunits. The preparation and evaluation of both enantiomersof CBI-CDPI₂ (25), CBI-CDPI, (26), CBI-indole₂ (27), CBI-indole₁ (28),and CBI-TMI (29) and their corresponding seco precursors 30-34 have beendisclosed in our early studies and their detailed comparisons with bothenantiomers of CC-1065 or the duocarmycins described. More recently, 27,28, and CBI-PDE-I₂ have been disclosed by Aristoff and co-workers. Thecomparative cytotoxic activity of these prior agents prepared in ourstudies is summarized in FIG. 9 along with that of the correspondingCPI-based analog.

In an extension of our investigations which first revealed efficaciousantitumor activity for 27, we have expanded the studies to thepreparation and evaluation of 57-61, a larger series based on 27. TheDNA binding subunits of CC-1065 and the duocarmycins contribute inseveral ways to the properties of the natural products. They contributesignificantly to the DNA binding affinity which serves both to increasethe rate of DNA alkylation relative to 9 and to thermodynamicallystabilize the inherently reversible DNA alkylation reaction. While theformer has been suggested to be the origin of the differences in thecytotoxic potency of 1 and 11 by others based principally on thecomparisons of (+)-N-BOC-CPI (11), (+)-CPI-indole₁, and (+)-CPI-indole₂,we have proposed that it is the latter that constitutes the biologicallysignificant distinction. This thermodynamic versus kinetic distinctionwas first proposed before the reversibility of the DNA alkylationreaction was experimentally verified and was based in part on theobservation that the cytotoxic potency of a class of agents wouldplateau. For example, (+)-CC-1065, (+)-CPI-PDE-I₁, and (+)-CPI-CDPI_(n)(n=1-3) were found to be indistinguishable in our cytotoxic assays(IC₅₀=20 pM, L1210). Although the five agents exhibit large differencesin their rates of DNA alkylation, all five form thermodynamically stableadducts under physiological conditions. We attribute the increase incytotoxic potency of CPI-CDPI_(n) (n=1-3) vs 11 to noncovalent bindingstabilization of the reversible DNA adduct formation and that it is thesimple event not extent of this stabilization that results in theiressentially equivalent properties. This interpretation further suggeststhat CPI-indole₁ and CBI-indole₁ lack the sufficient stabilization forobservation of full potency. Moreover, the interpretation is consistentwith the observation that a maximum potency is achievable and that thelevel of this potency is directly related to the functional stability ofthe agents. Thus, the CBI-based agents examined to date exhibit asimilar plateau of potency (5 pM, L1210) but at a level 4× more potentthan that of the corresponding CPI-based agents (20 pM, L1210).

In addition, the DNA binding subunits of CC-1065 contribute to a strongAT-rich DNA binding selectivity which we have recently shown not onlycontributes to the alkylation selectivity of the agents but exerts anoverriding dominate control. In early studies, we were able todemonstrate that the noncovalent binding affinity was derived nearlyexclusively from stabilizing van der Waals contacts and hydrophobicbinding. Not only did the studies suggest that CC-1065 is bestrepresented as a selective alkylating agent superimposed on the trimerskeleton but removal of the peripheral methoxy and hydroxy substituents(PDE-I-CDPI) had no effect on its noncovalent AT-rich bindingselectivity and little effect on its binding affinity. This dependenceon hydrophobic binding stabilization results in preferential binding inthe narrower, deeper AT-rich regions of the minor groove where thestabilizing van der Waals contacts are maximal (ΔG°=9.5-11.5 kcal/mol).Moreover, such studies suggested seminal ways in which the DNA bindingsubunits could be simplified (removal of polar substituents) withoutaltering the characteristics responsible for the essential DNA bindingaffinity or selectivity.

The DNA binding subunits of the agents may also have a significantimpact on the physical properties and characteristics of the agents.Most apparent is the remarkable solubility properties of CC-1065 whichis essentially insoluble in all solvents except DMSO or DMF includingpolar protic or aprotic solvents, water, or nonpolar solvents. A majorimpact that structural variations in the central and right hand subunitsmay have is in the solubility properties of the agent and hence itsbiodistribution and bioavailability.

Finally, we have speculated that the extent of the noncovalent bindingstabilization of the inherently reversible DNA alkylation reaction maybe responsible for the unusual, delayed toxicity of CC-1065. That is,the extensive noncovalent binding stabilization of 1 that renders itsDNA alkylation reaction irreversible while that of simpler agentsincluding 2-3 are slowly reversible under physiological conditionsoffers a potential explanation for the apparently confusing toxicityprofile among the analogs detailed to date. The only agents that haveexhibited the delayed toxicity that we are aware of are (+)-CC-1065 (1),(+)-CPI-CDPI₂, and (+)-CBI-PDE-I₂. Each provide irreversible adductformation under physiological conditions, and the unnatural enantiomersof each, which form inherently less stable and more reversible adducts,do not exhibit the delayed toxicity. Although speculative, it doessuggest that simplified DNA binding subunits which provide sufficientbut not extensive binding stabilization of the reversible DNA adductmight offer important advantages that relate to the inherent repair orreversal of nonproductive DNA alkylation sites. Moreover, this wouldalso provide a further strong rationale for the use of less reactivealkylation subunits (CBI versus CPI) whose DNA adducts, while stable,are inherently less stable and more readily reversed.

The preparation of the expanded series of agents 57-61 and theircorresponding seco derivatives 52-56 is summarized in FIG. 10. Thesimplified DNA binding subunits were assembled by coupling methyl5-aminoindole-2-carboxylate (35) or methyl5-aminobenzoxazole-2-carboxylate (36) with 37-39. Hydrolysis of themethyl esters 40-45 (LiOH, THF-CH₃OH—H₂O, 25° C.) followed by couplingof the carboxylic acids 46-51 with freshly generated 16 (EDCI, DMF, 25°C.) deliberately conducted in the absence of added base providedexcellent yields of the seco agents 32 and 52-56. Spirocyclization of 32and 52-56 was effected by treatment with NaH, DBN, or P₄-tBu andprovided the agents 27 (CBI-indole₂) and 57-61.

The results of the cytotoxic evaluations of the agents are summarized inFIG. 11 along with those of CBI-indole₂ (27) and CPI-indole₂. Severalaspects of these comparative evaluations are notable. First, the naturalenantiomers are substantially more potent than the unnatural enantiomers(130-1000×). In addition, the seco agents 32 and 52-56 exhibited thesame levels of cytotoxic activity as the cyclopropane containing agentswhere compared although this was not investigated in detail. Mostnotably and with the exception of 60, the cytotoxic potency of naturalenantiomers of the new agents were equivalent to or exceeded those of 27and 57 and all were 2-6× more potent than the corresponding CPI analog.Moreover, the potencies of 32 and 52-56 approach or are equivalent withthe ceiling of potency observed with 25-36 (5 pM).

Although we have described an extensive account of the DNA alkylationproperties of both enantiomers of 25-27, 28, and 29 elsewhere, theircomparisons with the corresponding CPI-based agents and theirrelationship to the biological evaluations merit summarizing. In thesestudies, a detailed investigation leading to the definition of the 3.5-5base pair AT-rich adenine N3 alkylation selectivity of the agents weredisclosed for both the natural and unnatural enantiomers, models weredisclosed which accommodate the reversed binding orientations and offsetAT-rich alkylation selectivity, and a beautiful explanation emergedwhich explains the diminished DNA capabilities of the unnaturalenantiomers. Moreover, a clearer picutre of the origin of the DNAalkylation selectivity and the structural features of the agentsresponsible have emerged from these studies. In a detailed comparativeexamination of the DNA alkylation properties of the CBI-based agents andthe corresponding CPI-based analog or duocarmycin SA based agent, theyhave been found to exhibit identical DNA alkylation selectivities. Thisis nicely illustrated in FIGS. 3 and 4 with the comparisons ofCBI-indole₂ (27)/CPI-indole₂ (4) and CBI-TMI (29)/duocarmycin SA (2),respectively. In addition, the CBI-based agents have been shown toalkylate DNA both at a faster rate and with a greater efficiency thanthe corresponding CPI-based agent. This is nicely illustrated in FIG. 21with the comparison of (+)-CBI-indole₂ (27) and (+)-CPI-indole₂ (4)where 27 is 10× more efficient at alkylating w794 (4° C. or 37° C., datafor latter not shown). Moreover, when the relative rates of DNAalkylation were directly compared at the single high affinity site ofw794 DNA, that of CBI-indole₂ was considerably faster. k(27)/k(4)=14,FIGS. 23A-23B. In contrast, the natural enantiomer of CBI-based agentsand corresponding duocarmycin SA based agents have been found toalkylate w794 DNA with essentially indistinguishable efficiencies (FIG.22) and at comparable rates, k(29)/k(2)=0.9, FIGS. 23A-23B.

In addition, because of the unnatural enantiomer sensitivity todestabilizing steric interactions surrounding the duocarmycin C7, CPI C7or CBI C8 center, the unnatural enantiomers of the simpler CBI-basedanalogs are approximately 4-100× less effective than the naturalenantiomers. In comparison, the unnatural enantiomers of the CPI-basedanalogs are 10-1000× less effective and the duocarmycin SA based analogsor agents are 1-10× less effective in both the cytotoxic assays and intheir relative DNA alkylation rate or efficiency. Moreover, thisdistinction in the enantiomers diminishes only with the larger agents,ie. 25, where the extensive noncovalent binding interactions aresufficiently large to overcome the destabilizing steric interactions ofthe unnatural enantiomer alkylation. Importantly, these trends followclosely the relative cytotoxic potency of the agents, the relativestabilities of the three classes of agents, and highlight the enhanceddistinctions of the CBI- versus CPI-based analogs and the comparableproperties of the duocarmycin SA and CBI-based agents. Fundamental tomembers of this class of antitumor antibiotics, the natural enantiomersof the agents were found to follow a well-defined relationship betweensolvolysis (functional) stability (−log k, pH 3) and cytotoxic potency(1/log IC₅₀, L1210) where the chemically more stable agents within agiven class exert the greatest potency, FIGS. 24A-24E. FIGS. 24A-24Einclude data for 4-6 available classes of agents that bear fivedifferent DNA binding subunits which we have examined, and although thisrelationship is undoubtedly a second order polynomial indicative of aparabolic relationship that will exhibit an optimalstability-reactivity/potency, the agents employed in FIGS. 24A-24E liein a near linear range of such a plot. What is unmistakable in thecomparisons, is the fundamental direct correlation between functional(solvolytic) stability and cytotoxic potency.

CBI-CDPBO₁ and CBI-CDPBI₁: Deep-Seated Structural Variations in the DNABinding Subunits. The efforts of Lown and Dervan have demonstrated thatthe distamycin AT-rich noncovalent binding selectivity may be altered toaccommodate a G-C base-pair or to exhibit progressively altered AT-GCrich binding selectivity through introduction of a nitrogen within thebackbone core structure capable of serving as hydrogen bonding acceptor.Accordingly, we have investigated whether similar changes in the corestructure of CC-1065 would impact on its DNA binding selectivity andresulting DNA alkylation selectivity. Key to the importance of thisexamination was the recognition that the more rigid structure ofCC-1065, its rigid helical bound conformation, and its near exclusivedependence on stabilizing van der Waals contacts and hydrophobic bindingwhich dictates the preference for binding and alkylation within thenarrower, deeper AT-rich minor groove may not be so easily overridden byintroduction of a single hydrogen bond acceptor or donor.

In the conduct of these studies, we reported the preparation of (+)- andent-(−)-CBI-CDPBO₁ (62), (+)- and ent-(−)-CBI-CDPBI₁ (64) and theircorresponding seco precursors 63 and 65 bearing deep-seatedmodifications in the DNA binding subunit including the incorporation ofa nitrogen atom capable of functioning as a hydrogen bond acceptor(CDPBO, CDPBI) or hydrogen bond donor (CDPBI) on their inside convexface which is projected to be in intimate contact with the minor groovefloor.

The initial comparisons were made with agents containing a single DNAbinding subunit where the single deep-seated structural modification inthe DNA binding subunit might be expected to exert a more pronouncedeffect. In these studies, the DNA alkylation selectivities andefficiencies of the natural enantiomers of 62 and 64 were found to beessentially identical. Moreover, both were approximately 100× lessefficient at alkylating DNA than (+)-CBI-CDPI₁ (26). Thus, the simpleincorporation of a single nitrogen into 64 versus 26 has a pronouncedand detrimental effect on the relative efficiency of DNA alkylation.Identical to trends detailed in our prior work on the CBI-derivedagents, the unnatural enantiomers of 62 abd 64 proved to be 10-100× lessefficient at alkylating DNA than the corresponding natural enantiomers.

More interesting was the observed DNA alkylation selectivities of 62 and64. The DNA alkylation selectivities of (+)-62 and (+)-64 wereessentially identical and both were comparable to the selectivityobserved with (+)-26. Although the DNA alkylation selectivity of (+)-62and (+)-64 potentially could have been significantly altered or havebecome increasingly more tolerant of a GC base-pair in the alkylationsequence, the selectivity proved more revealing than this simpleexpectation. Not only did (+)-62 and (+)-64 alkylate DNA with the nearidentical selectivity of (+)-26, but the unnatural enantiomerselectivity for 62 and 64 proved essentially identical to that ofent-(−)-26. Thus, in a manner essentially identical to (+)- andent-(−)-26 which exhibit distinct alkylation selectivities(5′-A/TA/TA/TA versus 5′-A/TAA/TA/T, respectively) characteristic of thereverse binding orientations and offset 3.5 base-pair AT-rich bindingsites surrounding the alkylation site, the two entantiomers of 62 and 64alkylated essentially the same sites as the corresponding enantiomers of26 within duplex DNA. Moreover, this was observed to occur not with theincreasing tolerance for incorporation of GC base-pairs in thealkylation sequence, but rather with a diminished DNA alkylationefficiency (100×) relative to that of (+)- and ent-(−)-CBI-CDPI₁ (26).The potential origin of these effects have been discussed elsewhere.

The cytotoxic properties of 62-65 and that of the closely related CBIagents are summarized in FIG. 13. Consistent with their relativeefficiencies of DNA alkylation, the natural enantiomers of 62 and 64were essentially indistinguishable (500-1000 pM, L1210) and 100-200×less potent than (+)-CBI-CDPI₁ (26). Thus, the introduction of thesingle nitrogen atom in the DNA binding subunit of 64 reduced thebiological potency 100 to 200-fold. Consistent with prior observations,the natural enantiomers of 62 and 64 were 10-100× more potent than thecorresponding unnatural enantiomers.

CBI-Indole-NMe₃ ⁺: Electropositive Substituents Capable of Enhancing DNAAlkylation Efficiency Through Stabilizing Electrostatic Interactions. Inrecent studies, we have studied the impact that electronegative andelectropositive substituents placed on peripheral face of the agentshave on the noncovalent DNA binding affinity and selectivity. In thesestudies, we defined a destabilizing contribution to the DNA bindingaffinity that results from the introduction of a strong electronegativesubstituent and described a substantial enhancement of noncovalentbinding affinity that results from introduction of an electropositivesubstituent. This was attributed to a spatially well-defineddestabilizing or stabilizing electrostatic interaction with thenegatively charged DNA phosphate backbone, respectively, and was foundto have little impact on the intrinsic AT-rich binding selectivity ofthe parent agents. These studies were recently extended to thepreparation of 66-68, close analogs of 28/33, containing a peripheralquaternary ammonium salt capable of providing a strong, stabilizingelectrostatic interaction with the DNA phosphate backbone. Consistentwith expectations, the agents 66-68 alkylated DNA with the same relativeefficiency as 1-2 and were approximately 100× more effective than 28 or33 which lack the ammonium salt substituent. Because of the smaller sizeof the agents, they exhibited a DNA alkylation selectivity that wassubtly altered from that of (+)-CC-1065, but comparable to that of(+)-duocarmycin SA. In addition, the agents were water soluble and offerpotential advantages over the existing agents.

Consequently, we were interested in the relative cytotoxic properties of66-68 and the results of their evaluations are summarized in FIG. 15.Although 66-68 were essentially identical in their cytotoxic potencies(10 nM), they proved to be slightly less potent than (+)-CBI-indole₁(28) and approximately 1000× less potent than (+)-1 and (+)-2. This isin contrast to expectations based on their relative efficiencies of DNAalkylation. Although this was not investigated, we attribute thisdiminished cytotoxic potency to ineffective cellular penetrationrequired for the agents to reach their intracellular target.

Additional Analogs. In the course of our investigations, severaladditional agents have been examined including 73 and 75, simplederivatives of the CBI alkylation subunit which possess enhanced DNAalkylation capabilities and in vitro cytotoxic potency by virtue ofstabilizing electrostatic DNA binding. That is, in place of the DNAbinding affinity derived from hydrophobic binding and stabilizing vander Waals contacts provided by the central and right-hand subunits of1-3, the simple electrostatic binding affinity provided by theprotonated amine of 73 and 75 with the negatively charged phosphatebackbone of DNA proved sufficient to substantially enhance the DNAalkylation intensity and in vitro cytotoxic activity.

The semicarbazide of CBI and its seco chloride precursor were preparedas detailed in FIG. 16. Treatment of bis(2,4-dinitrophenyl)carbonate(69) with tert-butylcarbazate (70, 1 equiv, 24° C., 2 h, EtOAc) provided71 (61%) and a convenient acylating agent for introduction of thetert-butyloxycarbonyl protected hydrazide. N-deprotection of 15 (3 NHCl-EtOAc, 24° C., 20 min, 100%) followed by immediate treatment of theunstable amine hydrochloride salt 16 with 71 (1.3 equiv, 1 equiv Et₃N,24° C., 5.5 h, THF, 91%) provided 72 in excellent yield. Acid-catalyzedN-BOC deprotection of 72 provided 73 and exposure of 72 or 73 to 5%aqueous NaHCO₃-THF (24° C.) provided 74 or 75, respectively.

The results of the in vitro cytotoxic evaluation of the N-semicarbazideof CBI conducted on its more stable seco precursor 73 are detailed inFIG. 18 along with the comparative results from the evaluation ofN-BOC-CBI (15) and 72. Notably, 73 which possesses the free amineexhibited more potent in vitro cytotoxic activity than its precursorpossessing the tert-butylcarbazate (72, ca. 100×) or N-BOC-CBI (9)itself, and proved to be only 100× less potent than (+)-CC-1065.

Consistent with the trends observed in the relative cytotoxic potency ofthe agents, the intensity of DNA alkylation similarly increased with theintroduction of the free semicarbazide and the results of these studieshave been detailed elsewhere. Thus, the introduction of a positivelycharged functionality (protonated amine) onto the simple CBI alkylationsubunit served to enhance the DNA alkylation intensity of the agentpresumably by providing noncovalent electrostatic DNA binding affinityto the agents. Consistent with the enhancement in the DNA alkylationintensity (100×), the in vitro cytotoxic activity of the agentsincreased correspondingly (100×).

The introduction of a terminal semicarbazide onto CBI-CDPI₂ was carriedfor comparison purposes (FIG. 17). Acid-catalyzed deprotection ofN-BOC-CDPI₂ (76, CF₃CO₂H, 25° C., 1 h) followed by coupling of crudeamine salt with 71 (1.5 equiv, 1 equiv Et₃N, 25° C., 19 h, 91% overall)provided 77 in excellent conversion. Direct coupling of 77 with freshlygenerated 16 (3 equiv EDCI, DMF, 25° C., 10 h) provided 78 (65%) in goodconversions. Acid-catalyzed deprotection (3M HCl-EtOAc, 25° C., 30 min)cleanly provided 79 (95-100%).

The examination of 78 and 79 revealed that this alteration in theC-terminus of CBI-CDPI₂ (25) did not impact on the inherent propertiesof the agent, FIG. 18. Thus, in contrast to 73 where the introduction ofa stabilizing electrostatic interaction enhances the DNA alkylationefficiency and cytotoxic potency of the agent, it had no impact on theproperties of 79 versus 78/25. Presumably, this may be attributed to thefact that the noncovalent hydrophobic binding affinity of 25 is alreadysufficient to provide full stabilization of the reversible DNA adductand the maximal cytotoxic potency and that the additional electrostaticstabilization provided in 79 is unnecessary.

Notably, the terminal acyl hydrazides of 73, 75 and 79 may serve asuseful functionality for subsequent reversible or irreversibleconjugation with tumor selective delivery systems and such studies areunderway.

In contrast to early speculation, deep-seated modifications in theCC-1065 and duocarmycin alkylation subunit are well tolerated and theCBI-based analogs proved to be potent cytotoxic agents and efficaciousantitumor compounds. A direct relationship between functional stabilityand cytotoxic potency was defined and validated. As such, the readilyaccessible CBI-based analogs were found to be 4× more stable and 4× morepotent than the corresponding analogs containing the CPI alkylationsubunit of CC-1065 and comparable in potency to the agents containingthe duocarmycin SA alkylation subunit. Similarly, the CBI-based agentsalkylate DNA with an unaltered sequence selectivity at an enhanced rateand with a greater efficiency than the corresponding CPI analogs andwere comparable to the corresponding DSA analog. Systematic modificationand simplification of the attached DNA binding subunits have provided aseries of synthetic and potent cytotoxic agents including 25-29 and57-61 whose biological profile are under further study. A number of theagents detailed herein exhibit potent and efficacious antitumoractivity.

One aspect of the invention is directed to a compound represented by thefollowing structure:

wherein R₁ is selected from the group consisting of —CH₂CH₃ (alkyl),—NHCH₃ (—N-alkyl), —OCH₃ (O-alkyl), —NH₂, —NHNH₂, —NHNHCO₂ ^(t)Bu, and aradical. The radical is represented by the following structure:

wherein A is selected from the group consisting of NH and O; B isselected from the group consisting of C and N; R₂ is selected from thegroup consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl(C1-C6)₃ and a first N-substituted pyrrolidine ring; R₃ is selected fromthe group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl(C1-C6)₃, the first N-substituted pyrrolidine ring; R₄ is selected fromthe groupconsisting of hydrogen, hydroxyl, O-alkyl (C1-C6), and N-alkyl(C1-C6)₃; R₅ is selected from the group consisting of hydrogen,hydroxyl, O-alkyl (C1-C6), and N-alkyl (C1-C6)₃; and V₁ represents afirst vinylene group between R₂ and R₃. The following provisos apply: ifR₂ participates in the first N-substituted pyrrolidine ring, then R₃also particlates in the first N-substituted pyrrolidine ring; if R₃participates in the first N-substituted pyrrolidine ring, then R₂ alsoparticlates in the first N-substituted pyrrolidine ring; if R₂ and R₃participate in the first N-substituted pyrrolidine ring, then R₄ and R₅are hydrogen; and if R₂ is hydrogen, then R₄ and R₅ are hydrogen and R₃is N-alkyl (C1-C6)₃. The first N-substituted pyrrolidine ring is fusedto the first vinylene group between R₂ and R₃ and is represented by thefollowing structure:

wherein V₁ represents the first vinylene group between R₂ and R₃; R₆ isselected from the group consisting of —CH₂CH₃ (alkyl), —NHCH₃(—N-alkyl), —OCH₃ (O-alkyl), —NH₂, —NHNH₂, —NHNHCO₂ ^(t)Bu, and aradical. The radical is represented by the following structure:

wherein C is selected from the group consisting of NH and O; D isselected from the group consisting of C and N; R₇ is selected from thegroup consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl(C1-C6)₃, and a second N-substituted pyrrolidine ring; R₈ is selectedfrom the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6),N-alkyl (C1-C6)₃, the second N-substituted pyrrolidine ring; and V₂represents the second vinylene group between R₇ and R₈. The followingprovisos apply: if R₇ participates in the N-substituted pyrrolidinering, then R₈ also particlates in the N-substituted pyrrolidine ring;and if R₈ participates in the N-substituted pyrrolidine ring only if R₇also particlates in the N-substituted pyrrolidine ring. The secondN-substituted pyrrolidine ring is fused to the second vinylene groupbetween R₇ and R₈ and is represented by the following structure:

wherein V₂ represents the second vinylene group between R₇ and R₈; andR₉ is selected from the group consisting of —CH₂CH₃ (alkyl), —NHCH₃(—N-alkyl), —OCH₃ (O-alkyl), —NH₂, —NHNH₂, and —NHNHCO₂ ^(t)Bu.

Another aspect of the invention is directed to a compound represented bythe following structure:

wherein X is selected from the group consisting of chlorine, bromine,iodine, and OTOS; and R₁ is selected from the group consisting of—CH₂CH₃ (alkyl), —NHCH₃ (—N-alkyl), —OCH₃ (O-alkyl), —NH₂, —NHNH₂,—NHNHCO₂ ^(t)Bu, and a radical. The radical is represented by thefollowing structure:

wherein A is selected from the group consisting of NH and O; B isselected from the group consisting of C and N; R₂ is selected from thegroup consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl(C1-C6)₃ and a first N-substituted pyrrolidine ring; R₃ is selected fromthe group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl(C1-C6)₃, the first N-substituted pyrrolidine ring; R₄ is selected fromthe groupconsisting of hydrogen, hydroxyl, O-alkyl (C1-C6), and N-alkyl(C1-C6)₃; R₅ is selected from the group consisting of hydrogen,hydroxyl, O-alkyl (C1-C6), and N-alkyl (C1-C6)₃; and V₁ represents afirst vinylene group between R₂ and R₃. The following provisos apply: ifR₂ participates in the first N-substituted pyrrolidine ring, then R₃also particlates in the first N-substituted pyrrolidine ring; if R₃participates in the first N-substituted pyrrolidine ring, then R₂ alsoparticlates in the first N-substituted pyrrolidine ring; if R₂ and R₃participate in the first N-substituted pyrrolidine ring, then R₄ and R₅are hydrogen; and if R₂ is hydrogen, then R₄ and R₅ are hydrogen and R₃is N-alkyl (C1-C6)₃. The first N-substituted pyrrolidine ring is fusedto the first vinylene group between R₂ and R₃ and is represented by thefollowing structure:

wherein V₁ represents the first vinylene group between R₂ and R₃; R₆ isselected from the group consisting of —CH₂CH₃ (alkyl), —NHCH₃(—N-alkyl), —OCH₃ (O-alkyl), —NH₂, —NHNH₂, —NHNHCO₂ ^(t)Bu, and aradical. The radical is represented by the following structure:

wherein C is selected from the group consisting of NH and O; D isselected from the group consisting of C and N; R₇ is selected from thegroup consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl(C1-C6)₃, and a second N-substituted pyrrolidine ring; R₈ is selectedfrom the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6),N-alkyl (C1-C6)₃, the second N-substituted pyrrolidine ring; and V₂represents the second vinylene group between R₇ and R₈. The followingprovisos apply: if R₇ participates in the N-substituted pyrrolidinering, then R₈ also particlates in the N-substituted pyrrolidine ring;and if R₈ participates in the N-substituted pyrrolidine ring only if R₇also particlates in the N-substituted pyrrolidine ring. The secondN-substituted pyrrolidine ring is fused to the second vinylene groupbetween R₇ and R₈ and is represented by the following structure:

wherein V₂ represents the second vinylene group between R₇ and R₈; andR₉ is selected from the group consisting of —CH₂CH₃ (alkyl), —NHCH₃(—N-alkyl), —OCH₃ (O-alkyl), —NH₂, —NHNH₂, and —NHNHCO₂ ^(t)Bu.

Another aspect of the invention is directed to a compound represented bythe following structure:

wherein A is selected from the group consisting of NE and O and B isselected from the group consisting of NH, O, and S.

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

Another aspect of the invention is directed to a compound compoundrepresented by the following structures:

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

where R is selected from the group comprising of: H, 5-NMe₃ ⁺, 6-NMe₃ ⁺,7-NMe₃ ⁺.

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

where R is selected from the group comprising of: CO₂ ^(t)Bu, H—HCl.

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

Another aspect of the invention is directed to, a compound compoundrepresented by the following structure:

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

where R is selected from the group comprising of: H—HCl, CONHMe, CO₂CH₃,COEt.

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

wherein A is selected from the group consisting of O and R is selectedfrom the group consisting of NO₂ and NH₂.

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

wherein B is selected from the group consisting of O and S.

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

wherein A is selected from the group consisting of NH and O and B isselected from the group consisting of NH, O, and S and R is selectedfrom the group consisting of H and CH₃.

Another aspect of the invention is directed to a compound compoundrepresented by the following structure:

wherein A is selected from the group consisting of NH and O and B isselected from the group consisting of NH, O, and S.

DESCRIPTION OF FIGURES

FIG. 1 illustrates the structures of (+)-CC-1065 (+)-1b, (+)-duocarmycinSA (+)-2, (+)-duocarmycin A and (+)-3.

FIG. 2 illustrates the structures of adozelesin (derivatives 4 and 5),carzelesin (6) and KW-2189 (8).

FIG. 3 illustrates the structures of CBI analogs which are based on CPIanalogs.

FIG. 4 illustrates the synthesis of compound 14 with the indicatedintermediates, substrates, and intermediate steps.

FIGS. 5A-5B illustrate the synthesis of compounds 16, 20, 22, 24, 26,28, 30, 32 and 34 with the indicated intermediates, substrates, andintermediate steps. FIG. 6 illustrates the structures of compounds 9,10, 11, 12, and 14 with the indicated rate constant k (s⁻¹, pH 3) ofacid-catalyzed solvolysis, half life t_(1/2) in solution and cytotoxicactivity IC₅₀ (L1210 cells)

FIG. 7 illustrates a summary of the cytotoxic activity (IC₅₀ (L1210,nM)) of the set of agents examined and the comparison with the relatedCPI-based agents. Agents are indicated as natural or unnatural.

FIG. 8 illustraes the structures of CBI analogs 25-29 and theintermediate CBI analogs 30-34 with the indicated R groups.

FIG. 9 illustrates a summary of the comparative cytotoxic activity (IC₅₀(L1210, nM)) of prior agents prepared in our studies. Agents areindicated as natural or unnatural.

FIG. 10 illustrates the synthesis of compounds 82, 84, 86, 88, 90 and 92with the indicated intermediates, substrates, and intermediate steps.

FIG. 11 illustrates a summary of the results of the cytotoxicevaluations of the agents examined and the comparison with the relatedCPI-based agents. Agents are indicated as natural or unnatural.

FIG. 12 illustrates the structures of CBI analogs (+)-62, 63, (+)-64 and65, and indicates functional groups on the CDPBO and CDPBI analogs whichare H-bond acceptor and donor, respectively.

FIG. 13 illustrates a summary of the results of the cytotoxicevaluations (IC₅₀ (L1210, pM))of the agents examined and the comparisonwith the related CPI-based agents. Agents are indicated as natural orunnatural.

FIG. 14 illustrates the structures of CBI intermediate analogs 33, 66,67 and 68.

FIG. 15 illustrates a summary of the relative alkylation efficiency (RelDNA Alkylation) and representative cytotoxicity (IC₅₀ (L1210, nM) ofagents 66-68 which contain a peripheral quaternary ammonium salt and areclose analogs with 28 (values indicated)

FIG. 16 illustrates the synthesis of compounds 102 and 104 with theindicated intermediates, substrates, and intermediate steps.

FIG. 17 illustrates the synthesis of compound 112 with the indicatedintermediates, substrates, and intermediate steps.

FIG. 18 illustrates a summary of the relative alkylation efficiency (RelDNA Alkylation) and representative cytotoxicity (IC₅₀ (L1210, nM)) ofagents 9, 72, 73, 78, 79, and 25.

FIGS. 19A-19B illustrate the solvolysis of 23. Top: UV-visible spectraof 23 in 50% CH₃OH-aqueous buffer (pH 3) recorded at various timeintervals (0, 21, 57, 84, 160, 371 h). Bottom: Plot of the disappearanceof 23, 1-[(A-A_(i))/(A_(f)-A_(i)) versus time from which the first ordersolvolysis rate constant was derived.

FIGS. 20A-20D illustrate the results of studies acid-catalyzedsolvolysis studies of 21-24 conducted at pH 3 (CH₃OH—H₂O) were followedspectrophotometrically by UV with the disappearance of thecharacteristic long-wavelength absorption band of the CBI chromophoreand with the appearance of a short-wavelength absorption bandattributable to the seco-N-BOC-CBI derivative.

The comparisons of 21-24 reveal a direct, linear relationship betweenthe cytotoxic potency (L1210, log 1/IC₅₀) and the solvolytic stability(−log k_(solv), pH 3) of the agents. Similarly, a linear relationshipwas found between the electron-withdrawing properties of the N²substituents (Hammett_(p) constant) and the solvolysis reactivity (−logk_(solv), pH 3) of the agents with the strongest electron-withdrawingsubstituents providing the most stable agents. This latter relationshipreflects the influence of the N² substituent on the ease of C4 carbonylprotonation required for catalysis of solvolysis and cyclopropyl ringcleavage with the stronger electron-withdrawing N² substituentsexhibiting slower solvolysis rates. Less obvious but more fundamental,the observations were found to follow a predictable linear relationshipbetween the cytotoxic potency (L1210, log 1/IC₅₀) and theelectron-withdrawing properties of the N² substituent (Hammett_(p)) withthe strongest electron-withdrawing substituents providing thebiologically most potent agents.

FIG. 21 illustrates the thermally-induced strand cleavage ofdouble-stranded DNA (144 bp, nucleotide no. 138-5238, clone w794) after24 h incubation of agent-DNA at 4° C. followed by removal of unboundagent and 30 inutes incubation at 100° C.; denaturing 8% polyacrylamidegel and autoradiography. Lanes 1-4, Sanger G,C,A, and T sequencingreactions; lane 5, control labeled w794 DNA; lanes 6-8, (+)-CPI-indole₂((+)-4, 1×10⁻⁴−1×10⁻⁶ M); lanes 9-11, (+)-CBI-indole₂ ((+)-27,1×10⁻⁵−1×10⁻⁷ M).

FIG. 22 illustrates the thermally-induced strand clealvage of 5′end-labeled duplex DNA (clone w794, 144 bp, nucleotide no 138-5238).Incubation of agent-DNA at 25° C. (24 h) followed by removal of unboundagent and 30 min thermolysis at 100° C., denaturing 8% PAGE, andautoradiography. Lanes 1-2, ent-(−)-duocarmycin SA ((−)-2, 1×10⁻⁶ and1×10⁻⁷ M); lanes 3-5, (+)-duocarmycin SA, ((+)-2, 1×10⁻⁶−1×10⁻⁸ M);lanes 6-9, G, C, A and T sequencing reactions; lane 10, control labeledw794 DNA; lanes 11-13, (+)-CBI-TMI ((+)-29, 1×10⁻⁶−1×10⁻¹ M); lanes14-15, ent-(−)-CBI-TMI ((−)-29, 1×10⁻⁵ and 1×10⁻⁶ M).

FIGS. 23A-23B represent the following: Top: Plot of % integrated opticaldensity (% IOD) versus time established through autoradiography of 5′³²P end-labeled DNA and used to monitor the relative rate of w794alkylation at the 5′-AATTA high affinity site for (+)-CBI-indole₂ (27)and (+)-CPI-indole₂ (4); 37° C., 0-5 d, 1×10⁻⁵ M agent. Bottom: Plot of% integrated optical density (% IOD) versus time established throughautoradiography of 5′ ³²P end-labeled DNA and used to monitor therelative rate of w794 alkylation at the 5′-AATTA high affinity site for(+)-duocarmycin SA (2) and (+)-CBI-TMI (29); 4° C., 0-24 h, 1×10⁻⁶ Magent.

FIGS. 24A-24E illustrate the data for 4-6 available classes of agentsthat bear five different DNA binding subunits which we have examined andalthough this relationship is undoubtedly a second order polynomialindicative of a parabolic relationship that will exhibit an optimalstability-reactivity/potency, the agents employed in the figure lie in anear linear range of such a plot. What is unmistakable in thecomparisons, is the fundamental direct correlation between functional(solvolytic) stability and cytotoxic potency.

FIG. 25 illustrates the synthesis of compound 128 with the indicatedintermediates, substrates, and intermediate steps.

FIG. 26 illustrates the synthesis of compound 154 with the indicatedintermediates, substrates, and intermediate steps.

FIG. 27 illustrates the synthesis of compounds 160 and 162 with theindicated intermediates, substrates, and intermediate steps.

FIGS. 28A-28B illustrate the structures of CBI analogs (+)-160,intermediate 156, (+)-162 and intermediate 158, and indicates functionalgroups on the CDPBO and CDPBI analogs which are H-bond acceptor anddonor, respectively.

FIG. 29 illustrates the synthesis of compounds 182, 184 and 186 with theindicated intermediates, substrates, and intermediate steps.

FIG. 30 illustrates the synthesis of compounds 190, 192, 194, and 196with the indicated intermediates, substrates, and intermediate steps.

SYNTHETIC METHODS

Preparation ofN-(tert-Butyloxycarbonyl)-4-benzyloxy-1-iodo-2-naphthyl-amine (4).

Compound 4 (Illustrated in FIG. 4). A solution of 2 (as prepared inthree steps from commercially available 1,3-dihydroxynaphthalene (71%overall), by Boger et. al. J. Org. Chem. 1992, 2873) (1.28 g, 3.66 mmol)in 60 mL of a 1:1 mixture of tetrahydrofuran-CH₃OH was cooled to −78° C.and 20 μL of H₂SO₄ (or 20 mg P-toluenesulfonic acid H₂O) in 0.5 mL oftetrahydrofuran was added. N-Iodosuccinimide (910 mg, 4.03 mmol) in 5 mLof tetrahydrofuran was then introduced by cannula over 5 min. Uponcomplete reaction (ca. 3 h at −78° C.), 10 mL of saturated aqueousNaHCO₃ and 50 mL of Diethyl ether were added. The reaction mixture waswarmed to 25° C. and solid NaCl was added to saturate the aqueous layer.The organic layer was separated and the aqueous layer was extracted withDiethyl ether (2×10 mL). The organic layers were combined, washed withsaturated aqueous NaHCO₃ (1×10 mL) and saturated aqueous NaCl (2×10 mL),dried (Na₂SO₄), and concentrated. The crude product was purified byelution through a short column of SiO₂ (2×4 cm, 20% Ethylacetate-hexane)to provide 4 (1.48 g, 85%) as a white, crystalline solid: mp 111-112°C.; ¹H NMR (CDCl₃, 400 MHz) 8.21 (dd, 1H, J=8.4, 0.8 Hz), 8.03 (s, 1H),8.01 (d, 1H, J=7.6 Hz), 7.55-7.33 (m, 7H), 7.30 (br s, 1H), 5.27 (s,2H), 1.56 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) 155.7, 152.8, 138.3, 136.6,134.8, 131.2, 128.7, 128.6, 128.5, 128.1, 127.8, 124.5, 123.7, 122.7,100.0, 81.2, 70.4, 28.4; IR (film) 3384, 2974, 2923, 1739, 1617, 1598,1567, 1515, 1494, 1444, 1392, 1366, 1332, 1226, 1152, 1107, 1082 cm⁻¹;FABHRMS (NBA-CsI) m/z 607.9715 (C₂₂H₂₂INO₃+Cs⁺ requires 607.9699). Anal.Calcd for C₂₂H₂₂INO₃: C, 55.59; H, 4.67; N, 2.95. Found: C, 55.85; H,4.43; N, 2.97.

Preparation of2-[N-(tert-Butyloxycarbonyl)-N-(2-propenyl)]amino-4-benzyloxy-1-iodonaphthalene(6) (Illustrated in FIG. 4) A solution of 4 (1.38 g, 2.90 mmol) in 25 mLof dimethylformamide at 0° C. was treated with NaH (60% dispersion inoil, 139 mg, 3.5 mmol) in several portions over 15 min. After 45 min,allyl bromide (1.05 g, 8.70 mmol) was added and the reaction mixture waswarmed to 25° C. and stirred for 3 h. The reaction mixture was quenchedby addition of 20 mL saturated aqueous NaHCO₃ and the aqucous layer wasextracted with Ethylacetate (4×15 mL). The combined organic layers werewashed with saturated aqueous NaCl (2×10 mL), dried (Na₂SO₄), andconcentrated under reduced pressure. Centrifugal thinlayerchromatography(2 mm Chromatotron plate, 20-50% CH₂Cl₂-hexanes) provided 6 (1.24 g,83%, typically 80-95%) as a colorless oil (mixture of amide rotamers inCDCl₃): ¹H NMR (CDCl₃, 400 MHz) (major rotamer) 8.30 (d, 1H, J=8.2 Hz),7.29 (d, 1H, J=8.2 Hz), 7.60-7.29 (m, 7H), 6.67 (s, 1H), 5.96-5.86 (m,1H), 5.27-4.96 (m, 4H), 4.52 (dd, 1H, J=15.0, 5.7 Hz), 3.79 (dd, 1H,J=15.0, 7.2 Hz, 1.29 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) (major rotamer)154.9, 153.8, 143.0, 136.4, 135.3, 133.5, 132.7, 128.7, 128.6, 128.4,128.1, 127.2, 126.1, 122.4, 117.9, 108.0, 95.0, 80.3, 70.2, 52.1, 28.3;IR (film) 3048, 2976, 2923, 1703, 1590, 1403, 1367. 1326, 1251, 1147,1105 cm⁻¹; FABHRMS (NBA-NaI) m/z 538.0855 (C₂₅H₂₆INO₃+Na⁺ requires538.0860).

Preparation of5-(Benzyloxy)-3-(tert-butyloxycarbonyl)-1-(2′,2′,6′,6′-tetramethylpiperidinyl-N-oxymethyl)-1,2-dihydro-3H-benz[e]indole(8) (Illustrated in FIG. 4) A solution of 6 (1.85 g, 3.59 mmol) andTempo (1.68 g, 10.8 mmol) in 120 mL of freshyl distilled benzene(Na/benzophenone) under N₂ was treated with Bu₃SnH (1.045 g, 3.59 mmol).The solution was warmed at 70° C. and three additional equivalents ofTempo (3×0.56 g) and Bu₃SnH (4×1.045 g) were added sequentially in fourportions over the next 45 min. After 1 h, the solution was cooled to 25°C. and the volatiles were removed under reduced pressure. Centrifugalthinlayerchromatography (4 mm Chromatotron plate, 0-10%Ethylacetate-hexanes gradient elution) followed by recrystallizationfrom hexanes provided 8 (1.71 g, 87%, typically 70-90%) as whiteneedles: mp 170-172° C.; ¹H NMR (C₆D₆, 400 MHz) 8.56 (d, 1H, J=8.3 Hz),8.38 (br s, 1H), 7.77 (d, 1H, J=8.4 Hz), 7.35 (ddd, 1H, J=8.3, 7.6, 1.2Hz), 7.28 (d, 2H, J=7.0 Hz), 7.20 (t, 1H, J=7.6 Hz), 7.15 (t, 2H), 7.07(t, 1H, J=7.2 Hz), 4.36 (m, 1H), 4.12 (dd, 1H, J=9.0, 4.5 Hz), 3.84 (m,1H), 3.61 (m, 1H), 1.53 (s, 9H), 1.37-1.17 (m, 6H), 1.22 (s, 3H), 1.13(s, 3H), 1.07 (s, 3H), 1.00 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz) 154.1,151.4, 140.0, 135.8, 129.4, 127.3, 126.7, 126.3, 125.8, 121.9, 121.6,121.4, 121.1, 114.8, 95.3, 79.2, 69.0, 58.5, 51.5, 38.4, 38.3, 37.2,31.9, 27.3, 18.9, 15.8; IR (film) 2973, 2930, 1704, 1626, 1582, 1460,1406, 1381, 1367, 1328, 1266, 1144, 10,7, 787, 759 cm⁻¹; FABHRMS(NBA-CsI) m/z 677.2340 (C₃₄H₄₄N₂O₄+Cs⁺ requires 677.2355). Anal Calcdfor C₃₄H₄₄N₂O₄: C, 74.97; H, 8.14; N, 5.14. Found: C, 74.68; H, 8.37; N,5.19.

Preparation of5-(Benzyloxy)-3-(tert-butyloxycarbonyl)-1-(hydroxy-methyl)-1,2-dihydro-3H-benz[e]indole(10) (Illustrated in FIG. 4) A solution of 8 (1.61 g, 2.95 mmol) in 70mL of a 3:1:1 mixture of HOAc-tetrahydrofuran-H₂O was treated with zincpowder (2.31 g, 35.4 g atoms) and the resulting suspension was warmed at70° C. with vigorous stirring. After 2 h, the reaction mixture wascooled to 25° C. and the zinc was removed by filtration. The volatileswere removed under reduced pressure and the resulting residue wasdissolved in 40 mL of Ethylacetate and filtered. The solution wasconcentrated and subjected to centrifugal thinlayerchromatography (4 mmChromatotron plate, 15-35% Ethylacetate-hexanes gradient elution) toprovide 10 (0.96 g, 1.19 g theoretical, 80%) identical in all respectswith authentic material (see Boger et. al J. Am. Chem. Soc. 1992, 114,5487.

Preparation of compound 12 (Illustrated in FIG. 4) A solution of 10 (1.0equiv.) and triphenylphosphine (2.0 equiv.) in methylene chloride (0.2Molar) at 24° C. under argon was treated with carbon tetrachloride (6.0equiv.) and the reaction mixture was stirred for 10 h (24° C.). Flashchromatography and resolution as derived from the coorespondingmandelate ester (see Boger et. al J. Org. Chem., 1990, 55, 5830, expt.31), affords the enantiomerically pure compound 12.

Preparation of compound 14 (Illustrated in FIG. 4) A solution of 12 (1.0equiv.) in 0.05 Molar tetrahydrofuran at 0° C. under argon was treatedsequentially with a 25% aqueous ammonium formate (0.5 M) and 10%palladium/carbon (0.10 equiv.) and the reaction mixture stirredvigorously for 2.5 h (0° C.). Ether was mixed with the reaction mixture,and the mixture was dried (Magnesium sulphate). The solid was removed byfiltration through Celite (ether wash). Concentration of the filtrate invacuo afforded 14 as colorless needles.

Preparation of compound 16 (Illustrated in FIGS. 5A-5B) Compound 14 (1.0equiv.) was treated with anhydrous 3N hydrochloric acid in ethyl acetate(0.033 M) at 24° C. for 20 min. The solvent was removed in vacuo toafford crude, unstable 16 (95%-100%). The crude product was directlycarried on without purification (for a related procedure see Boger et.al. J. Org. Chem. 1990, 23, 5831 experimental 5).

Preparation of compound 18 (Illustrated in FIG. 5A-5B) A solution of 14(1.0 equiv.) in 0.01 M of tetrahydrofuran-dimethylformamide (1:1) wascooled to 0° C. and treated with NaH (1.5 equiv). The reaction mixturewas slowly warmed to 24° C. and stirred for 3.5 h. The mixture wasplaced on a flash chromatography column (SiO₂, 0.5×3 mm), and elutedwith 5-10% CH₃OH—CHCl₃ (gradient elution) to afford 18 as a brightyellow solid.

Preparation of compound 20 (Illustrated in FIGS. 5A-5B) A solution of 16(1.0 equiv.) in tetrahydrofuran (0.02 M) was treated with 0.02 M of 5%aqueous NaHCO₃ and the two-phase mixture was stirred at 24° C. for 5 hunder N₂. The reaction mixture was extracted with Ethylacetate (3×). Theorganic layer was dried (Na₂SO₄) and concentrated. Flash chromatographyafforded 20 as a pale yellow solid. As an alternative, one can obtaincompound 20 from compound 18 in trifluoroacetic acid (0.1 equiv.) andmethylene chloride (0.02 M) at 0° C. for 2 hours (for a relatedprocedure see Boger et. al. J. Org. Chem. 1990, 23, 5831 experimental5).

Preparation of1-(Chloromethyl)-5-hydroxy-3-[(methylaminocarbonyl]-1,2-dihydro-3H-benz[e]indole(22) (Illustrated in FIGS. 5A-5B) Phenol 14 (6.9 mg, 21 μmol) wastreated with anhydrous 3M HCl-Ethylacetate at 24° C. for 30 min underAr. The solvent was removed in vacuo to afford crude, unstable 16(quantitative). A solution of 16 and NaHCO₃ (5.2 mg, 62 μmol, 3 equiv)in tetrahydrofuran (0.3 mL) was cooled to 0° C. and treated with CH₃NCO(2.4 μL, 41 μmol. 2 equiv). The reaction mixture was kept at 0° C. for 1h under Ar before the solvent was removed under a stream of N₂. Flashchromatography (SiO₂, 0.5×3 cm, 50-80% Ethylacetate-hexane gradientelution) afforded 22 (5.0 mg, 6.0 mg theoretical, 83%) as a palegreenish solid: ¹H NMR (CD₃OD, 400 MHz) 8.11 (d, 1H, J=8.4 Hz, C6-H),7.68 (s, 1H, C4-H), 7.65 (d, 1H, J=8.4 Hz, C9-H), 7.44 (t, 1H, J=8.2 Hz,C8-H), 7.24 (t, 1H, J=8.3 Hz, C7-H), 4.06-4.14 (m, 3H, C2-H₂, C1-H),3.92 (d, 1H, J=11.4 Hz, CHHCl) 3.51-3.53 (m, 1H, CHHCl), 2.83 (s, 3H,CH₃); IR (film)_(max) 3816, 1624, 1585, 1522, 1384, 1339, 1250, 1121cm⁻¹; FABHRMS (NBA) m/e 290.0818 (M⁺+H, C₁₅H₁₅ClN₂O₂ requires 290.0822).Natural (1S)-22: [α]⁵−4.5 (c 0.36, CH₃OH). Ent-(1R)-22: [α]⁵+5.7 (c 0.11CH₃OH).

Preparation ofN²-[(Methylamino)carbonyl]-1,2,9,9a-tetrahydro-cyclo-propa-[c]benz[e]-indol-4-one(24) (Illustrated in FIGS. 5A-5B) A solution of 22 (3.0 mg, 10.3 μmol)in dimethylformamide (0.9 mL) was cooled to 0° C. and treated with1,8-DIZABICYCLO[5.4.0]UNDEC-7-ENE (3.2 μL, 21 μmol, 2 equiv) and themixture was stirred at 4° C. for 2 d. The solvent was removed in vacuoand flash chromatography (SiO₂, 0.5×3 cm, 0-10% CH₃OH-Ethylacetategradient elution) afforded 24 (2.3 mg, 2.6 mg theoretical, 90%) as apale yellow solid: ¹H NMR (CD₃OD, 400 MHz) 8.08 (d, 1H, J=8.0 Hz, C5-H),7.55 (t, 1H, J=7.7 Hz, C7-H), 7.40 (t, 1H, J=8.0 Hz, C6-H), 7.08 (d, 1H,J=7.7 Hz, C8-H), 6.93 (s, 1H, C3-H), 4.05 (dd, 1H, J=10.0, 5.1 Hz,C1-H), 3.95 (d, 1H, J=10.0 Hz, C1-H), 3.08 (m, 1H, C9a-H), 2.79 (s, 3H,CH₃), 1.73 (dd, 1H, J=7.8, 4.2 Hz, C9-H), 1.48 (t, 1H, J=4.6 Hz, C9-H);IR (neat)_(max) 3358, 2920, 1680, 1622, 1594, 1539, 1466, 1458, 1410,1281 cm⁻¹; UV (CH₃OH)_(max) 311 (15000), 257 (7300), 217 (17000), 200(17000) nm; UV (tetrahydrofuran)_(max) 304 (11000), 248 (7200), 216(17000), 208 (15000) nm; FABHRMS (NBA) m/e 255.1140 (M⁺+H, C₁₅H₁₄N₂O₄requires 255.1134). Natural (+)-24: [α]³+183 (c 0.08, CH₃OH).Ent-(−)-24: [α]⁵−184 (c 0.13, CH₃OH).

Preparation of1-(Chloromethyl)-5-hydroxy-3-(methoxycarbonyl)-1,2-dihydro-3H-benz[e]indole(26) (Illustrated in FIGS. 5A-5B). A solution of freshly prepared, crude16 (52 μmol) and NaHCO₃ (13.2 mg, 157 μmol. 3 equiv) in tetrahydrofuran(0.5 mL) was cooled to 0° C. and treated with ClCO₂CH₃ (8.1 μL, 104μmol. 2 equiv). The reaction mixture was warmed to 25° C. and stirredfor 1.5 h before it was concentrated in vacuo. Flash chromatography(SiO₂, 1×10 cm, 20-40% Ethylacetate-hexane gradient elution) afforded 26(15 mg, 15 mg theoretical, 100%) as a white solid: ¹H NMR (CDCl₃, 400MHz) 8.59 (br s, 1H, OH), 8.25 (d, 1H, J=8.1 Hz, C6-H), 7.94 (br s, 1H,C4-H), 7.62 (d, 1H, J=8.3 Hz, C9-H), 7.50 (t, 1H, J=8.1 Hz, C8-H), 7.35(t, 1H, J=8.2 Hz, C7-H), 4.31 (d, 1H, J=11.4 Hz, C2-H), 4.12 (apparentt, 1H, J=9.3 Hz, C2-H), 3.90-3.99 (m, 5H, C1-H, CHHCl, CO₂CH₃), 3.40 (t,1H, J=10.5 Hz, CHHCl); IR (film)_(max) 3275, 2918, 1678, 1442, 1388,1335 cm⁻¹; FABHRMS (NBA) m/e 291.0664 (M⁺, C₁₅H₁₄ClNO₄ requires291.0662). Natural-(1S)-26: [α]³−30.3 (c 0.11, CH₃OH). Ent-(1R)-26:[α]³+31.8 (c 0.24, CH₃OH).

Preparation ofN²-(Methoxycarbonyl)-1,2,9,9a-tetrahydro-cyclopropa-[c]benz[e]-indol-4-one(28) (Illustrated in FIGS. 5A-5B). A solution of 26 (10.0 mg, 34 μmol)in tetrahydrofuran (3 mL) was cooled to 0° C. and treated with1,8-DIZABICYCLO[5.4.0]UNDEC-7-ENE (10.5 μL, 68 μmol, 2 equiv). Thereaction mixture was stirred at 4° C. for 41 h and then warmed to 24° C.and stirred for 10 h.⁶¹ The reaction mixture was treated with saturatedaqueous NH₄Cl (3 mL) and extracted with CH₂Cl₂ (3×2 mL). The combinedorganic layer was dried (Na₂SO₄) and concentrated. Flash chromatography(SiO₂, 1×10 cm, 10-50% Ethylacetate-hexane gradient elution) afforded 28(8.1 mg, 8.7 mg theoretical, 87%) as a yellow solid: ¹H NMR (CDCl₃, 400MHz) 8.21 (d, 1H, J=7.8 Hz, C5-H), 7.48 (t, 1H, J=7.6 Hz, C7-H), 7.39(t, 1H, J=7.8 Hz, C6-H), 6.87 (s, 1H, C3-H), 6.86 (d, 1H, J=7.6 Hz,C8-H), 3.86-4.08 (m, 2H, CH₂N), 3.86 (s, 3H, CH₃), 2.79 (m, 1H, C9a-H),1.56-1.59 (m, 1H, C9-H), 1.39 (t, 1H, J=4.8 Hz, C9-H); IR (neat)_(max)3283, 2984, 1728. 1626, 1559, 1436, 1405, 1380, 1328, 1277, 1246, 1195,1118, 1077, 1021, 764 cm⁻¹; UV(CH₃OH)_(max) 307 (32000), 255 (24000),216 (32000), 200 (33000) nm; UV (tetrahydrofuran)_(max) 296 (33000), 253(23000), 217 (38000), 203 (44000) nm; FABHRMS:(NBA) m/e 256.0986 (M⁻+H,C₁₅H₁₃NO₃ requires 256.0974). Natural (+)-28: [α]³+198 (c 0.48, CH₃OH).Ent-(−)-28: [α]⁵−196 (c 0.14, CH₃OH).

Preparation of1-(Chloromethyl)-5-hydroxy-3-propionyl-1,2-dihydro-3H-benz[e]indole (30)(Illustrated in FIGS. 5A-5B). A solution of freshly prepared, crude 16(45 μmol) and NaHCO₃ (11.3 mg, 135 μmol, 3 equiv) in tetrahydrofuran(0.4 mL) was cooled to 0° C. and treated with ClCOEt (8 μL, 90 μmol, 2equiv). The reaction mixture was warmed to 24° C. and stirred for 5 hunder N₂. The solvent was removed under a stream of N₂. Flashchromatography (SiO₂, 1×10 cm, 10-40% Ethylacetate-hexane gradientelution) afforded 30 (12.8 mg, 13 mg theoretical, 98%) as a white solid:¹H NMR (CDCl₃, 400 MHz) 9.70 (br s, 1H, OH), 8.39 (s, 1H, C4-H), 8.32(d, 1H, J=8.0 Hz, C6-H), 7.66 (d, 1H, J=8.3 Hz, C9-H), 7.52 (t, 1H,J=8.3 Hz, C8-H), 7.39 (t, 1H, J=8.3 Hz, C7-H), 4.32 (dd, 1H, J=2.0, 10.9Hz, C2-H), 4.23 (d, 1H, J=10.8 Hz, C2-H), 4.04 (m, 1H, C1-H), 3.97 (dd,1H, J=2.9, 11.3 Hz, CHHCl), 3.41 (t, 1H, J=10.8 Hz, CHHCl), 2.59-2.72(m, 2H, CH₂CH₃), 1.39 (t, 3H, J=7.4 Hz, CH₂CH₃); IR (film)_(max) 3170,2918,1628, 1582, 1427, 1389 cm⁻¹; FABHRMS (NBA) m/e 290.0953 (M⁺+H,C₁₆H₁₆ClNO₂ requires 290.0953). Natural (1S)-30: [α]⁵−54 (c 0.08,tetrahydrofuran). Ent-(1R)-30: [α]⁵+59 (c 0.13, tetrahydrofuran).

Preparation ofN²-(Propionyl)-1,2,9,9a-tetrahydro-cyclopropa[c]benz[e]-indol-4-one (32)(Illustrated in FIGS. 5A-5B). A solution of 30 (5.0 mg, 17 μmol) intetrahydrofuran (0.9 mL) was treated with 0.9 mL of 5% aqueous NaHCO₃and the two-phase mixture was stirred at 24° C. for 5 h under N₂. Thereaction mixture was extracted with Ethylacetate (3×3 mL). The organiclayer was dried (Na₂SO₄) and concentrated. Flash chromatography(Florisil, 1×5 cm, 60% Ethylacetate-hexane) afforded 32 (4.2 mg, 4.3 mgtheoretical, 97%) as a pale yellow solid: ¹H NMR (CDCl₃, 400 MHz) 8.22(d, 1H, J=7.8 Hz, C5-H), 7.51 (t, 1H, J=7.5 Hz, C7-H), 7.40 (t, 1H,J=7.9 Hz, C6-H), 6.89 (br s, 1H, C3-H), 6.88 (d, 1H, J=7.8 Hz, C8-H),4.13-4.16 (m, 1H, C1-H), 4.03 (dd, 1H, J=10.6, 4.9 Hz, C1-H), 2.76-2.81(m, 1H, C9a-H), 2.54-2.56 (m, 2H, CH₂CH₃), 1.67 (dd, 1H, J=7.6 Hz, 4.5Hz, C9-H), 1.43 (t, 1H, J=4.8 Hz, C9-H), 1.22 (t, 3H, J=7.3 Hz, CH₃); IR(neat)_(max) 2924, 1698, 1626, 1599, 1562, 1461, 1406, 1241 cm⁻¹; UV(CH₃OH)_(max) 311 (16000), 258 (9100), 218 (14000), 201 (19000) nm; UV(tetrahydrofuran)_(max) 301 (15000), 253 (9400), 219 (15000), 204(15000) nm; FABHRMS (NBA) m/e 254.1173 (M⁺+H, C₁₆H₁₅NO₂ requires254.1181).Natural (+)-32: +193 (c 0.03, CH₃OH). Ent-(−)-32: −197 (c0.12, CH₃OH). Preparation ofN²-(Ethylsulfonyl)-1,2,9,9a-tetrahydro-cyclopropa[c]-benz[e]indol-4-one(34) (Illustrated in FIGS. 5A-5B). NaH (1.5 mg, 60% oil dispersion, 38μmol, 2.5 equiv) in a flame-dried flask was treated with (+)-CBI (20,3.0 mg, 15.2 μmol) in tetrahydrofuran (0.8 mL) and the mixture wasstirred for 10 min at 24° C. under N₂. A premixed solution ofTriethylamine (7 μL, 50 μmol, 3.3 equiv) and ClSO₂Et (10 μL, 106 μmol, 7equiv) in tetrahydrofuran (0.8 mL) was added and the reaction mixturewas stirred at 24° C. for 3 h before being concentrated. Flashchromatography (SiO₂, 0.5×3 cm, 40-60% Ethylacetate-hexane gradientelution) afforded 34 (2.0 mg, 4.4 mg theoretical, 45%) as a pale yellowsolid: ¹H NMR (CDCl₃, 400 MHz) 8.19 (d, 1H, J=7.8 Hz, C5-H), 7.49 (t,1H, J=8.3 Hz, C7-H), 7.39 (t, 1H, J=7.8 Hz, C6-H), 6.85 (d, 1H, J=7.8Hz, C8-H), 6.46 (s, 1H, C3-H), 4.09 (m, 2H, CH₂N), 3.21-3.28 (m, 2H,CH₂CH₃), 2.83 (m, 1H, C9a-H), 1.69 (dd, 1H, J=7.8, 4.6 Hz, C9-H), 1.54(t, 1H, partially obscured by H₂O, C9-H), 1.43 (t, 3H, J=7.4 Hz, CH₃);IR (neat)_(max) 2923, 1618, 1559, 1354, 1149 cm⁻¹; UV (CH₃OH)_(max) 301(12000), 248 (11000), 214 (15000) nm; UV (tetrahydrofuran)_(max) 293(13000), 248 (14000), 216 (16000), 208 (14000) nm; FABHRMS (NBA-NaI) m/e290.0850 (M⁺+H, C₁₅H₁₅NO₃S requires 290.0851). Natural (+)-34: +73 (c0.10, CHCl₃). Ent-(−)-34: −70 (c 0.12, CHCl₃).

Preparation of Solvolytic Reactivity of 24,28,32,34 (Illustrated inFIGS. 5A-5B). The compounds 24,28,32,34 were dissolved in CH₃OH (1.5mL). The CH₃OH solution was mixed with aqueous buffer (pH=3, 1.5 mL).The buffer contained 4:1:20 (v/v/v) 0.1 M citric acid, 0.2 M Na₂HPO₄,and H₂O, respectively. After mixing, the solvolysis solutions werestoppered and kept at 25° C. in the dark. The UV spectrum of thesolutions was measured 3-4 times in the first two days and twice a dayfor 2-4 weeks for 24,28,32 and 3 months for 34. The UV monitoring wascontinued until no further change was detectable. The long-wavelengthabsorption at 316 nm (24,28,32) or 306 nm (34) and short-wavelengthabsorption at 256 nm (24,28,34) or 248 nm (32) were monitored. Thesolvolysis rate constant and half-life were calculated from the datarecorded at the short wavelength (256 nm for 24,28,32 and 248 nm for 34)from the least square treatment (r=0.995, 24; r=0.997, 28; r=0.985, 32;r=0.994, 34) of the slopes of plots of time versus1-[(A-A_(initial))/A_(final)-A_(initial))].

Preparation of Methyl 5-Nitrobenzofuran-2-carboxylate (Illustrated inFIG. 10) 5-Nitrobenzofuran-2-carboxylic acid from Transworld chemicalsinc. (500 mg, 2.4 mmol) in 20 mL of CH₃OH was treated with 5 drops ofH₂SO₄. The reaction mixture was stirred at 24° C. for 24 h and warmed at50° C. for 2 h. The mixture was cooled to 24° C., diluted with H₂O (20mL) and saturated aqueous NaHCO₃ (20 mL), and extracted withEthylacetate (3×30 mL). The combined organic phase was dried (Na₂SO₄)and concentrated in vacuo. Flash chromatography (SiO₂, 2×20 cm, 40-60%Ethylacetate-hexane) afforded the methyl ester (469 mg, 534 mgtheoretical, 88%) as a white solid: mp >230° C. (dec); ¹H NMR (CDCl₃,400 MHz) 8.64 (d, 1H, J=2.3 Hz, C4-H), 8.37 (dd, 1H, J=2.3, 9.2 Hz,C6-H), 7.69 (d, 1H, J=9.1 Hz, C7-H), 7.64 (s, 1H, C3-H), 4.02 (s, 3H,CH₃); ¹³C NMR (CDCl₃-CD₃OD, 100 MHz) 157.8 (C), 122.7 (CH), 120.9 (C),119.4 (CH), 118.5 (C), 114.1 (CH), 112.7 (CH), 112.0 (C), 109.2 (C),52.4 (CH₃); IR (film)_(max) 3383, 3108, 1731, 1620, 1571, 1521, 1441,1349, 1270, 1177, 827, 750 cm⁻¹; FABHRMS (NBA) m/e 222.0405 (M⁺+H,C₁₀H₇NO₅ requires 222.0402).

Preparation of compound Methyl 5-Nitrobenzoimidazol-2-carboxylate (36)(Blustrated in FIG. 10). Condensation of 3-nitrobenzaldehyde with methyl2-azidoacetate (8 equiv, 6 equiv NaOCH₃, CH₃OH, −23 to 0° C., 6 h, 88%)follow by thermolysis of the resulting methyl 2-azidocinnamate (xylene,reflux, 4.5 h, 81%) provided a readily separable mixture (4:1) of methyl5- and 7-nitroindole-2-carboxylate. For methyl5-nitroindole-2-carboxylate: ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)12.65 (br s, 1H, NH), 8.73 (d, 1H, J=2.3 Hz, C4-H), 8.14 (dd, 1H, J=2.0,8.0 Hz, C6-H), 7.60 (d, 1H, J=8.0 Hz, C7-H), 7.45 (d, 1H, J=0.7 Hz,C3-H), 3.90 (s, 3H, CO₂CH₃); IR (film)_(max) 3316, 1701, 1614, 1531,1435, 1343, 1261, 1203, 992, 746 cm⁻¹. Catalytic hydrogenation of the5-NO₂ group (1 atm H₂, 0.1 wt equiv 10% Pd-C, Ethylacetate, 25° C., 4-5h) provided the corresponding amine. (36): 92%, mp 150-152° C. (CH₂Cl₂);¹H NMR (CDCl₃, 400 MHz) 8.72 (br s, 1H, NH), 7.23 (d, 1H, J=8.6 Hz,C7-H), 7.03 (dd, 1H, J=1.0, 2.1 Hz, C3-H), 6.93 (dd, 1H, J=1.0, 2.0 Hz,C4-H), 6.81 (dd, 1H, J=2.0, 8.6 Hz, C6-H), 3.93 (s, 3H, CO₂CH₃), 3.57(br s, 2H, NH₂); ¹³C NMR (CDCl₃, 100 MHz) 160.0 (C), 150.3 (C), 145.6(C), 143.0 (C), 127.7 (C), 117.7 (CH), 113.5 (CH), 112.6 (CH), 106.1(CH), 52.2 (CH₃); IR (film)_(max) 3320, 1691, 1628, 1531, 1437, 1376,1337, 1232, 1034, 997, 766 cm⁻¹; FABHRMS (NBA) m/e 190.0746 (M⁺+H,C₁₀H₁₀N₂O₂ requires 190.0742).

Preparation of Methyl 5-Aminobenzofuran-2-carboxylate (38) (Illustratedin FIG. 10). A solution of methyl 5-nitrobenzofuran-2-carboxylate (469mg, 2.12 mmol) in 50 mL of Ethylacetate was treated with 10% Pd—C (235mg, 0.5 wt equiv), placed under 1 atm of H₂, and stirred at 25° C. (12h). The catalyst was removed by filtration through Celite, and thesolvent was removed in vacuo. Flash chromatography (SiO₂, 2×20 cm,40-60% Ethylacetate-hexane) afforded 38 (360 mg, 404 mg theoretical,89%) as a pale yellow solid: mp 109-111° C. (CH₂Cl₂, pale yellow fineneedles); ¹H NMR (CDCl₃, 400 MHz) 7.36 (s, 1H, C3-H), 7.36 (d, 1H, J=8.1Hz, C7-H), 6.89 (d, 1H, J=2.4 Hz, C4-H), 6.83 (dd, 1H J=2.4, 8.9 Hz,C6-H), 3.94 (s, 3H, CH₃), 3.45 (br s, 2H, NH₂); IR (film)_(max) 3359,1725, 1562, 1488, 1434, 1331, 1301, 1222, 1158 cm⁻¹; FABHRMS (NBA) m/e192.0663 (M⁺+H, C₁₀H₉NO₃ requires 192.0661).

General Procedure for the Preparation of 46,48,50,52,54,56 (Illustratedin FIG. 10).

Methyl 5-aminoindole-2-carboxylate (36), or methyl5-aminobenzofuran-2-carboxylate (38),1-(3-DIMETHYLAMINOPROPYL)-3-ETHYLCARBODIIMIDE HYDROCHLORIDE (EDCI) (3equiv) and indole-2-carboxylic acid (40) Aldrich company,benzofuran-2-carboxylic acid (42) Aldrich company orbenzo[b]thiophene-2-carboxylic acid (44) Aldrich company (1 equiv) werestirred in dimethylformamide (0.04-0.06 M) at 24° C. under Ar for 12 h.The solvent was removed in vacuo, and the dry residue was mixed with H₂Oand stirred for 30 min. The precipitate was collected by centrifugationand washed with 1N aqueous HCl, saturated aqueous NaHCO₃, and H₂O.Drying the solid in vacuo afforded desired aqueous methyl esters46,48,50,52,54,56 in typical yields of 50-73%.

Methyl 5-[((1H-Indol-2′-yl)carbonyl)amino]-1H-indole-2-carbox-ylate (46)5 h, 61%; mp >270° C. (dec); ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)11.92 (s, 1H, NH), 11.69 (s, 1H, NH), 10.16 (s, 1H, NH), 8.16 (d, 1H,J=1.6 Hz, C4-H), 7.67 (d, 1H, J=8.0 Hz, C4′-H), 7.60 (dd, 1H, J=2.0, 8.9Hz, C6-H), 7.47 (d, 1H, J=8.3 Hz, C7′-H), 7.45 (d, 1H, J=9.0 Hz, C7-H),7.41 (s, 1H, C3-H), 7.21 (t, 1H, J=8.2 Hz, C6′-H), 7.18 (s, 1H, C3′-H),7.07 (t, 1H, J=7.1 Hz, C5′-H), 3.88 (s, 3H, CH₃); IR (neat)_(max) 3277,1700, 1652, 1553, 1535, 1310, 1247, 1225, 1022, 999, 742 cm⁻¹; FABHRMS(NBA-NaI) m/e 356.1004 (M⁺+Na, C₁₉H₁₅N₃O₃ requires 356.1011).

Methyl 5-[((Benzofuro-2′-yl)carbonyl)amino]-1H-indole-2-carboxylate(48): 5 h, 73%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)11.91 (br s, 1H, NH), 10.48 (br s, 1H, NH), 8.18 (d, 1H, J=1.8 Hz,C4-H), 7.83 (d, 1H, J=7.8 Hz, C4′-H), 7.76 (s, 1H, C3′-H), 7.72 (d, 1H,J=8.4 Hz, C7′-H), 7.61 (dd, 1H, J=1.9, 8.9 Hz, C6-H), 7.50 (t, 1H, J=8.4Hz, C6′-H), 7.44 (d, 1H, J=8.8 Hz, C7-H), 7.37 (t, 1H, J=7.6 Hz, C5′-H),7.18 (s, 1H, C3-H), 3.88 (s, 3H, CH₃); IR (film)_(max) 3333, 1695, 1658,1591, 1535, 1442, 1303, 1255, 746 cm⁻¹; FABHRMS (NBA) m/e 335.1036((M⁺+H, C₁₉H₁₄N₂O₄ requires 335.1032).

Methyl 5-[((Benzo[b]thieno-2′-yl)carbonyl)amino]-1H-indole-2-carboxylate(50): 7 h, 62%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)11.93 (br s, 1H, NH), 10.47 (br s, 1H, NH), 8.35 (s, 1H, C3′-H), 8.13(d, 1H, J=1.9 Hz, C4-H), 8.05 (d, 1H, J=7.0 Hz, C7′-H), 7.99 (d, 1H,J=6.7 Hz, C4′-H), 7.57 (dd, 1H, J=2.0, 8.9 Hz, C6-H), 7.44-7.50 (m, 2H,C6′-H, C5′-H), 7.44 (d, 1H, J=9.0 Hz, C7-H), 7.17 (s, 1H, C3-H), 3.87(s, 3H, CH₃); ¹³C NMR (DIMETHYLSULFOXIDE-d₆, 100 MHz) 161.7, 160.1,140.5, 140.4, 139.2, 134.6, 131.6, 127.7, 126.6, 126.4, 125.3 (two CH),125.0, 122.8, 119.9, 113.2, 112.6, 107.9, 51.8; IR (film)_(max) 3336,1694, 1633, 1532, 1455, 1336, 1309, 1257, 1232 cm⁻¹; FABHRMS (NBA) m/e351.0810 (M⁺+H, C₁₉H₁₄N₂O₃S requires 351.0803).

Methyl 5-[((1H-Indol-2′-yl)carbonyl)amino]benzofuran-2-carboxylate (52):8 h, 52%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz) 11.75 (s,1H, NH), 10.36 (s, 1H, NH), 8.34 (d, 1H, J=2.1 Hz, C4-H), 7.84 (dd, 1H,J=2.1, 9.0 Hz, C6-H), 7.83 (s, 1H, C3-H), 7.73 (d, 1H, J=9.0 Hz, C7-H),7.68 (d, 1H, J=8.0 Hz, C4′-H), 7.47 (d, 1H, J=8.3 Hz, C7′-H), 7.43 (s,1H, C3′-H), 7.22 (t, 1H, J=7.1 Hz, C6′-H), 7.07 (t, 1H, J=7.1 Hz,C5′-H), 3.89 (s, 3H, CH₃); IR (film)_(max) 3346, 1712, 1643, 1577, 1543,1308, 1289, 1234, 745 cm⁻¹; FABHRMS (NBA) m/e 335.1040 (M⁺+H, C₁₉H₁₄N₂O₄requires 335.1032).

Methyl 5-[((Benzofuro-2′-yl)carbonyl)amino]benzofuran-2-carboxylate(54): 12 h, 62%; mp >230° C.; ¹H NMR (CDCl₃, 400 MHz) 8.44 (br s, 1H,NH), 8.28 (apparent t, 1H, J=1.3 Hz, C4-H), 7.69 (d, 1H, J=7.7 Hz,C4′-H), 7.60 (d, 1H, J=0.8 Hz, C3-H or C3′-H), 7.54-7.56 (apparent d,3H, J=8.4 Hz), 7.51 (s, 1H, C3-H or C3′-H), 7.45 (t, 1H, J=7.2 Hz,C6′-H), 7.31 (t, 1H, J=7.9 Hz, C5′-H), 3.97 (s, 3H, CH₃); ¹³C NMR(CDCl₃, 100 MHz) 159.8 (C), 156.7 (C), 154.8 (C), 152.8 (C), 148.3 (C),146.3 (C), 133.3 (C), 127.6 (C), 127.4 (C), 127.3 (CH), 124.0 (CH),122.9 (CH), 121.0 (CH), 114.1 (CH), 114.0 (CH), 112.7 (CH), 111.8 (CH),111.7 (CH), 52.5 (CH₃); IR (film)_(max) 3382, 1729, 1663, 1562, 1541,1475, 1431, 1291, 1204, 1151, 1103 cm⁻¹; FABHRMS (NBA) m/e 336.0878(M⁺+H, C₁₉H₁₃NO₃ requires 336.0872).

Methyl 5-[((Benzo[b]thieno-2′-yl)carbonyl)amino]benzofuran-2-carboxylate(56): 8 h, 50%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)10.67 (s, 1H, NH), 8.38 (s, 1H, C3′-H), 8.31 (d, 1H, J=2.0 Hz, C4-H),8.06 (dd, 1H, J=1.7, 6.9 Hz, C7′-H), 8.00 (dd, 1H, J=1.8, 6.9 Hz,C4′-H), 7.83 (s, 1H, C3-H), 7.80 (dd, 1H, J=2.1, 9.0 Hz, C6-H), 7.74 (d,1H, J=9.0 Hz, C7-H), 7.50 (dt, 1H, J=1.7, 7.1 Hz, C6′-H), 7.47 (dt,J=2.0, 7.1 Hz, C5′-H), 3.89 (s, 3H, CH₃); IR (film)_(max) 3287, 1728,1657, 1546, 1473, 1436, 1296, 1216, 1154 cm⁻¹; FABHRMS (NBA) m/e352.0650 (M⁺+H, C₁₉H₁₃NO₄S, requires 352.0644).

General Procedure for the Preparation of 58,60,62,64,66,68 (Illustratedin FIG. 10).

A solution of one the methyl esters 46,48,50,52,54 or 56 prepared asabove in tetrahydrofuran-CH₃OH—H₂O (3:1:1) was treated with 4 equiv ofLiOH H₂O. The reaction mixture was stirred at 24° C. for 4-6 h. Thesolvent was removed and the dry residue was mixed with H₂O, acidifiedwith 1N aqueous HCl to pH 1. The precipitate was collected bycentrifugation and washed with H₂O (2×). Drying the solid in vacuoafforded the desired acid 58,60,62,64,66,68 with yields 80-100%.

5-[((1H-Indol-2′-yl)carbonyl)amino]-1H-indole-2-carboxylic Acid (58): 3h, 89%; mp >270° C. (dec); ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz) 11.82(s, 1H, NH), 11.23 (br s, 1H, NH), 10.14 (s, 1H, NH), 7.99 (s, 1H,C4-H), 7.66 (d, 1H, J=7.6 Hz, C4′-H), 7.48 (d, 1H, J=8.0 Hz, C6-H), 7.41(s, 1H, C3-H), 7.39-7.41 (m, 2H, C7′-H and C7-H), 7.20 (t, 1H, J=7.6 Hz,C6′-H), 7.06 (t, 1H, J=7.2 Hz, C5′-H), 6.69 (br s, 1H, C3′-H); IR(film)_(max) 3413, 3354, 3315, 1665, 1596, 1532, 1463, 1444, 1409, 1306,1222, 1159, 1080 cm⁻¹; FABHRMS (NBA) m/e 320.1041 (M⁺+H, C₁₈H₁₃N₃O₃requires 320.1035).

5-[((Benzofuro-2′-yl)carbonyl)amino]-1H-indole-2-carboxylic Acid (60): 5h, 77%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz) 11.62 (br s,1H, NH), 10.43 (s, 1H, NH), 8.12 (s, 1H, C4-H), 7.83 (d, 1H, J=7.6 Hz,C4′-H), 7.75 (s, 1H, C3′-H), 7.73 (d, 1H, J=8.4 Hz, C7′-H), 7.55 (d, 1H,J=9.2 Hz, C6-H), 7.50 (t, 1H, J=8.4 Hz, C6′-H), 7.40 (d, 1H, J=8.8 Hz,C7-H), 7.37 (t, 1H, J=7.2 Hz, C5′-H), 7.00 (br s, 1H, C3-H); IR(film)_(max) 3297, 1661, 1594, 1537, 1299, 1258, 1229, 743 cm⁻¹; FABHRMS(NBA) m/e 321.0880 (M⁺+H, C₁₈H₁₂N₂O₄ requires 321.0875).

5-[((Benzo[b]thieno-2′-yl)carbonyl)amino]-1H-indole-2-carboxylic Acid(62): 3 h; 80%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)11.63 (s, 1H, NH), 10.46 (s, 1H, NH), 8.36 (s, 1H, C3′-H), 8.08 (s, 1H,C4-H), 8.04 (d, 1H, J=6.8 Hz, C7′-H), 7.99 (d, 1H, J=6.6 Hz, C4′-H),7.52 (d, 1H, J=8.9 Hz, C6-H), 7.44-7.48 (m, 2H, C6′-H, C5′-H), 7.41 (d,1H, J=8.8 Hz, C7-H), 7.00 (s, 1H, C3-H); ¹³C NMR (DIMETHYLSULFOXIDE-d6,100 MHz) 163.1, 160.0, 140.6, 140.4, 139.3, 134.2, 131.1, 126.9, 126.3,125.3, 125.3 (CH and C), 125.0, 122.9, 118.9, 113.1, 112.4, 106.4; IR(film)_(max) 3429, 3375, 1648, 1542, 1431, 1305, 1249, 739 cm⁻¹; FABHRMS(NBA) m/e 337.0654 (M⁺+H, C₁₈H₁₂N₂O₃S requires 337.0647).

5-[((1H-Indol-2′-yl)carbonyl)amino]benzofuran-2-carboxylic Acid (64): 4h, 80%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz) 13.54 (br s,1H, CO₂H), 11.74 (s, 1H, NH), 10.34 (s, 1H, NH), 8.30 (d, 1H, J=1.8 Hz,C4-H), 7.81 (dd, 1H, J=1.9, 9.0 Hz, C6-H), 7.72 (s, 1H, C3-H), 7.70 (d,1H, J=7.6 Hz, C7-H), 7.68 (d, 1H, J=8.6 Hz, C4′-H), 7.47 (d, 1H, J=8.3Hz, C7′-H), 7.43 (s, 1H, C3′-H), 7.22 (t, 1H, J=8.0 Hz, C6′-H), 7.06 (t,1H, J=7.6 Hz, C5′-H); IR (film)_(max) 3285, 1703, 1649, 1547, 1475,1420, 1312, 1231, 1195, 1159, 744 cm⁻¹; FABHRMS (NBA) m/e 321.0870(M⁺+H, C₁₈H₁₂N₂O₄ requires 321.0875).

5-[((Benzofuro-2′-yl)carbonyl)amino]benzofuran-2-carboxylic Acid (66):12 h, 100%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz) 13.50(br s, 1H, CO₂H), 10.61 (s, 1H, NH), 8.22 (d, 1H, J=1.7 Hz, C4-H), 7.83(d, 1H, J=7.7 Hz, C4′-H), 7.78 (d, 1H, J=0.8 Hz, C3′-H), 7.73 (dd, 1H,J=0.7, 8.4 Hz, C7′-H), 7.72 (dd, 1H, J=2.1, 9.0 Hz, C6-H), 7.61 (d, 1H,J=8.9 Hz, C7-H), 7.50 (t, 1H, J=8.4 Hz, C6′-H), 7.37 (t, 1H, J=7.9 Hz,C5′-H), 7.35 (br s, 1H, C3-H); IR (film)_(max) 3362, 1709, 1659, 1564,1473, 1438, 1292, 1226, 1152, 790 cm⁻¹; FABHRMS (NBA) m/e 322.0720(M⁺+H, C₁₈H₁₁NO₅ requires 322.0715).

5-[((Benzo[b]thieno-2′-yl)carbonyl)amino]benzofuran-2-carboxylic Acid(68): 12 h, 82%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)13.61 br s, 1H, CO₂H), 10.67 (s, 1H, NH), 8.40 (s, 1H, C3′-H), 8.30 (d,1H, J=1.9 Hz, C4-H), 8.09 (dd, 1H, J=1.7, 6.8 Hz, C7′-H), 8.05 (dd, 1H,J=1.8, 6.2 Hz, C4′-H), 7.79 (dd, 1H, J=2.0, 9.1 Hz, C6-H), 7.73 (d, 1H,J=9.0 Hz, C7-H), 7.70 (br s, 1H, C3-H), 7.53 (dt, 1H, J=1.6, 7.1 Hz,C6′-H), 7.50 (dt, 1H, J=1.5, 7.1 Hz, C5′-H); IR (film)_(max) 3395, 1697,1653, 1551, 1479, 1296, 1273, 1231, 1155, 1024, 991, 762 cm⁻¹; FABHRMS(NBA) m/e 338.0480 (M⁺H, C₁₈H₁₁NO₄S requires 338.0487).

Preparation of compounds 70,72,74,76,78,80 (Illustrated in FIG.10)—General Procedure for the Coupling of 16 with 58,60,62,64,66,68.Phenol 14 was treated with anhydrous 3M HCl-Ethylacetate at 24° C. for30 min. The solvent was removed in vacuo to afford crude unstable 16(quantitative). A solution of 16, one of the carboxylic acids58,60,62,64,66,68 (1 equiv), and1-(3-DIMETHYLAMINOPROPYL)-3-ETHYLCARBODIIMIDE HYDROCHLORIDE (EDCI) (2-3equiv) in dimethylformamide (0.04-0.06 M) was stirred at 24° C. under N₂for 8-12 h. The reaction mixture was concentrated under vacuum andsuspended in H₂O and the precipitate was collected by centrifugation,and washed with H₂O (2×). Flash chromotagraphy (SiO₂, 40-60%tetrahydrofuran-hexane) afforded 70 and 72,74,76,78,80 in yields of60-90%.

3-[(5′-(((1H-Indol-2″-yl)carbonyl)amino)-1H-indol-2′-yl)carbonyl]-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole(70): 8.5 h, 73%; mp >255° C. (dec); ¹H NMR (dimethylforamide-d₇, 400MHz) 11.76 (s, 1H, NH), 11.69 (s, 1H, NH), 10.58 (s, 1H, NH), 10.28 (s,1H, OH), 8.40 (d, 1H, J=1.7 Hz, C4′-H), 8.25 (d, 1H, J=8.4 Hz, C6-H),8.10 (br s, 1H, C4-H), 7.96 (d, 1H, J=8.3 Hz, C4″-H), 7.73 (d, 1H,J=2.0, 8.9 Hz, C6′-H), 7.70 (d, 1H, J=8.0 Hz, C9-H), 7.61 (d, 1H, J=8.2Hz, C7″-H), 7.59 (d, 1H, J=8.8 Hz, C7′-H), 7.57 (t, 1H, J=8.1 Hz, C8-H),7.53 (s, 1H, C3′-H), 7.41 (t, 1H, J=8.0 Hz, C7-H), 7.30 (s, 1H, C3″-H),7.26 (t, 1H, J=8.0 Hz, C6″-H), 7.10 (t, 1H, J=7.9 Hz, C5″-H), 4.90(apparent t, 1H, J=10.8 Hz, C2-H), 4.76 (dd, 1H, J=1.8, 10.9 Hz, C2-H),4.30-4.34 (m, 1H, C1-H), 4.13 (dd, 1H, J=3.1, 11.0 Hz, CHHCl), 3.96 (dd,1H, J=7.8, 11.0 Hz, CHHCl); IR (film)_(max) 3258, 2923, 1659, 1624,1578, 1512, 1411, 1395, 1233, 745 cm⁻¹; FABHRMS (NBA) m/e 535.1526(M⁺+H, C₃₁H₂₃ClN₄O₃ requires 535.1537). Natural (1S)-70: [ ]⁵+70 (c0.17, dimethylformamide). Ent-(1R)-70: [α]⁵−70 (c 0.17,dimethylformamide).

3-[(5′-(((Benzofuro-2″-yl)carbonyl)amino)-1H-indol-2′-yl)carbonyl]-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole(72): 14 h, 60%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)11.73 (s, 1H, NH), 10.59 (s, 1H, NH), 10.56 (s, 1H, OH), 8.44 (d, 1H,J=1.0 Hz, C4′-H), 8.24 (d, 1H, J=8.4 Hz, C6-H), 8.10 (br s, 1H, C4-H),7.94 (d, 1H, J=8.3 Hz, C4″-H), 7.85 (d, 1H, J=7.8 Hz, C7″-H), 7.80 (dd,1H, J=2.0, 8.8 Hz, C6′-H), 7.78 (s, 1H, C3″-H), 7.68 (d, 1H, J=8.4 Hz,C9-H), 7.61 (d, 1H, J=8.6 Hz, C7′-H), 7.56 (t, 1H, J=7.0 Hz, C6″-H),7.52 (t, 1H, J=8.4 Hz, C8-H), 7.40 (t, 1H, J=7.8 Hz, C5″-H or C7-H),7.39 (t, 1H, J=7.7 Hz, C5″-H or C7-H), 7.32 (s, 1H, C3′-H), 4.90(apparent t, 1H, J=10.8 Hz, C2-H), 4.75 (dd, 1H, J=2.0, 10.8 Hz, C2-H),4.32-4.34 (m, 1H, C1-H), 4.13 (dd, 1H, J=3.2, 11.2 Hz, CHHCl), 3.96 (dd,1H, J=7.6, 11.2 Hz, CHHCl); ¹³C NMR (tetrahydrofuran-d₈, 100 MHz) 161.1(C), 156.9 (C), 155.9 (C), 155.7 (C), 151.2 (C), 143.6 (C), 134.8 (C),132.8 (C), 132.6 (C), 131.3 (C), 129.0 (two C), 128.0 (CH), 127.4 (CH),124.5 (CH), 124.4 (CH), 123.7 (C), 123.6 (CH), 123.4 (CH), 123.1 (CH),119.9 (CH), 116.0 (C), 114.1 (CH), 112.5 (CH), 112.4 (CH), 110.9 (CH),106.7 (CH), 101.4 (CH), 56.1 (CH), 47.2 (CH₂), 43.9 (CH₂); IR(film)_(max) 3272, 2954, 1610, 1585, 1513, 1408, 1253, 1135, 741 cm⁻¹;FABHRMS (NBA) m/e 536.1390 (M⁺+H, C₃₁H₂₂ClN₃O₄ requires 536.1377).Natural (1S)-72: [α]³+56 (c 0.23, tetrahydrofuran).

3-[(5′-(((Benzo[b]thieno-2″-yl)carbonyl)amino)-1H-indol-2′-yl)carbonyl]-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole(74): 11 h, 68%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)11.80 (s, 1H, NH), 10.52 (s, 1H, NH), 10.48 (s, 1H, OH), 8.40 (s, 1H,C3″-H), 8.20 (d, 1H, J=1.6 Hz, C4′-H), 8.15 (d, 1H, J=8.2 Hz, C6-H),8.08 (d, 1H, J=6.9 Hz, C7″-H), 8.03 (d, 1H, J=6.7 Hz, C4″-H), 8.01 (brs, 1H, C4-H), 7.88 (d, 1H, J=8.4 Hz, C9-H), 7.60 (dd, 1H, J=1.9, 8.9 Hz,C6′-H), 7.48-7.57 (m, 4H, C6″-H, C5″-H, C8-H, C7′-H), 7.39 (t, 1H, J=8.1Hz, C7-H), 7.25 (s, 1H, C3′-H), 4.85 (apparent t, 1H, J=10.8 Hz, C2-H),4.60 (dd, 1H, J=1.8, 11.0 Hz, C2-H), 4.24-4.28 (m, 1H, C1-H), 4.06 (dd,1H, J=3.1, 11.1 Hz, CHHCl), 3.91 (dd, 1H, J=7.3, 11.1 Hz, CHHCl); IR(film)_(max) 3286, 1655, 1628, 1587, 1518, 1409, 1262, 1239 cm⁻¹;FABHRMS (NBA) m/e 552.1152 (M⁺+H, C₃₁H₂₂ClN₃O₃S requires 552.1149).Natural (1S)-74: [α]³+111 (c 0.15, dimethylformamide).

3-[(5′-(((1H-Indol-2″-yl)carbonyl)amino)benzofuro-2′-yl)carbonyl]-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole(76): 13 h, 80%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)11.78 (s, 1H, NH), 10.51 (s, 1H, NH), 10.38 (s, 1H, OH), 8.35 (d, 1H,J=2.0 Hz, C4′-H), 8.12 (d, 1H, J=8.3 Hz, C6-H), 7.92 (br s, 1H, C4-H),7.86 (d, 1H, J=8.8 Hz, C9-H), 7.84 (dd, 1H, J=2.1, 9.0 Hz, C6′-H), 7.80(s, 1H, C3′-H), 7.76 (d, 1H, J=9.0 Hz, C7′-H), 7.69 (d, 1H, J=8.0 Hz,C4″-H), 7.53 (t, 1H, J=8.2 Hz, C8-H), 7.48 (d, 1H, J=8.4 Hz, C7″-H),7.45 (s, 1H, C3″-H), 7.38 (t, 1H, J=8.0 Hz, C7-H), 7.23 (t, 1H, J=8.0Hz, C6″-H), 7.07 (t, 1H, J=7.7 Hz, C5″-H), 4.79 (apparent t, 1H, J=9.8Hz, C2-H), 4.58 (d, 1H, J=9.9 Hz, C2-H), 4.24 (m, 1H, C1-H), 4.01 (dd,1H, J=3.1, 11.1 Hz, CHHCl), 3.89 (dd, 1H, J=7.4, 11.1 Hz, CHHCl); IR(film)_(max) 3274, 2928, 1655, 1621, 1579, 1546, 1414, 1390, 1329, 1240cm⁻¹; FABHRMS (NBA) m/e 536.1360 (M⁺+H, C₃₁H₂₂ClN₃O₄ requires 536.1377).Natural (1S)-76: [α]³+26 (c 0.36, dimethylformamide).

3-[(5′-(((Benzofuro-2″-yl)carbonyl)amino)benzofuro-2′-yl)carbonyl]-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole(78): 11 h, 88%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)10.72 (s, 1H, NH), 10.48 (s, 1H, OH), 8.38 (d, 1H, J=2.0 Hz, C4′-H),8.14 (d, 1H, J=8.3 Hz, C6-H), 7.71-7.95 (m, 7H), 7.54 (t, 1H, J=7.2 Hz,C8-H), 7.52 (t, 1H, J=7.4 Hz, C6″-H), 7.39 (t, 2H, J=7.9 Hz, C7-H andC5″-H), 4.79 (apparent t, 1H, J=10.6 Hz, C2-H), 4.59 (d, 1H, J=9.8 Hz,C2-H), 4.25 (m, 1H, C1-H), 4.02 (dd, 1H, J=3.0, 11.1 Hz, CHHCl), 3.90(dd, J=7.4, 11.1 Hz, CHHCl); IR (film)_(max) 3267, 2923, 1664, 1581,1554, 1410, 1390, 1328, 1256 cm⁻¹; FABHRMS (NBA) m/e 537.1210 (M⁺+H,C₃₁H₂₂ClN₂O₅ requires 537.1217). Natural (1S)-78: [α]³+26 (c 0.28,dimethylformamide).

3-[(5′-(((benzo[b]thieno-2″-yl)carbonyl)amino)benzofuro-2′-yl)carbonyl]-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole(80): 18 h, 68%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)10.69 (s, 1H, NH), 10.52 (s, 1H, OH), 8.40 (s, 1H, C3″-H), 8.32 (d, 1H,J=1.9 Hz, C4′-H), 8.13 (d, 1H, J=8.3 Hz, C6-H), 8.07 (d, 1H, J=8.7 Hz,C7″-H), 8.03 (d, 1H, J=6.2 Hz, C4″-H), 7.94 (br s, 1H, C4-H), 7.86 (d,1H, J=8.3 Hz, C9-H), 7.82 (dd, 1H, J=2.0, 9.0 Hz, C6′-H), 7.80 (s, 1H,C3′-H), 7.77 (d, 1H, J=9.0 Hz, C7′-H), 7.45-7.55 (m, 3H, C8-H, C6″-H,C5″-H), 7.38 (t, 1H, J=7.9 Hz, C7-H), 4.79 (apparent t, J=10.0 Hz,C2-H), 4.59 (d, 1H, J=10.0 Hz, C2-H), 4.22-4.26 (m, 1H, C1-H), 4.02 (dd,1H, J=3.0, 11.1 Hz, CHHCl), 3.88 (dd, 1H, J=7.4, 11.0 Hz, CHHCl); IR(film)_(max) 3259, 2923, 1659, 1630, 1583, 1549, 1413, 1392, 1336, 1244,1211 cm⁻¹; FABHRMS (NBA) m/e 553.0985 (M⁺+H, C₃₁H₂₁ClN₂O₄S requires553.0989). Natural (1S)-80: [α]³+30 (c 0.33, dimethylformamide).

Preparation of compounds 82 and 84,86,88,90,92 (Illustrated in FIG.10)—General Procedures for the Spirocyclization and Preparation of 82and 84,86,88,90,92. Method A: A suspension of NaH (60% oil dispersion, 2equiv) in tetrahydrofuran at 0° C. under Ar was treated with a solutionof 70, 72,74,76,78,80 prepared above intetrahydrofuran-dimethylformamide (1:1, ca. 0.015 M reactionconcentration). The reaction mixture was stirred at 0° C. for 30 min-1h. The solvent was removed in vacuo and the solid residue was washedwith H₂O and dried in vacuo. Flash chromatography (SiO₂, 50-70%tetrahydrofuran-hexane) afforded 82, 84,86,88,90,92 in 50-93% yield.

Method B: A solution of compounds 70, 72,74,76,78,80 intetrahydrofuran-dimethylformamide (2:1, ca. 0.015 M) was cooled to 0° C.and treated with 1,5-diazabicyclo[4.3.0]non-5-ene (DBN, 2 equiv). Thereaction-mixture then was allowed to warm to 24° C. and stirred for 2-4h. The solvent was removed in vacuo, and flash chromatography (SiO₂,50-70% tetrahydrofuran-hexane) afforded 82, 84,86,88,90,92 with 40-75%yield.

Method C: A sample of 70 (1.6 mg, 0.0030 mmol) in tetrahydrofuran (0.20mL) was treated with the phosphazene base P₄-t-Bu (3.3 μL, 1 M solutionin hexane, 1.1 equiv) at −78° C. The mixture was stirred under Ar at−78° C. for 40 min, at 0° C. for 6 h, and at 25° C. for 2 h. The crudemixture was purified directly by chromatography (SiO₂, 60%tetrahydrofuran-hexane) to provide 82 (1.4 mg, 1.5 mg theoretical, 93%)as a yellow solid.

N²-[5′-(((1H-Indol-2″-yl)carbonyl)amino)-1H-indol-2′-yl)carbonyl]-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one(82, CBI-indole₂): Method A, 93%; method B, 75%; method C, 93%; mp >240°C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 300 MHz) 11.86 (br s, 1H, NH), 11.73(br s, 1H, NH), 10.19 (s, 1H, NH), 8.24 (d, 1H, J=2.6 Hz, C4′-H), 8.02(d, 1H, J=8.0 Hz, C5-H), 7.67 (d, 1H, J=7.8 Hz, C4″-H), 7.63 (m, 2H,C6-H and C7-H), 7.47 (m, 4H), 7.29 (s, 1H, C3′-H or C3″-H), 7.25 (m, 2H,C8-H and C6″-H), 7.07 (t, 1H, J=7.3 Hz, C5″-H), 6.98 (s, 1H, C3-H), 4.65(dd, 1H, J=4.9, 10.2 Hz, C1-H), 4.53 (apparent d, 1H, J=10.2 Hz, C1-H),3.20 (m, 1H, obscured by H₂O, C9a-H), 1.77 (dd, 1H, J=4.2, 7.4 Hz,C9-H), 1.73 (t, 1H, J=4.2 Hz, C9-H); IR (KBr)_(max) 3432, 1648, 1522,1384, 1266, 1126, 744 cm⁻¹; UV (dimethylformamide)_(max) 316 (=45000),274 nm (25000); FABHRMS (NBA) m/e 499.1792 (M⁺+H, C₃₁H₂₂N₄O₃ requires499.1770). Natural (+)-82: [α]³+114 (c 0.03, dimethylformamide), [α]⁵+81(c 0.12, tetrahydrofuran). Ent (−)-82: [α]⁵−120 (c 0.1,dimethylformamide), [α]⁵−81 (c 0.12, tetrahydrofuran).

N²-[(5′-(((Benzofuro-2″-yl)carbonyl)amino)-1H-indol-2′-yl)carbonyl]-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one(84): Method A, 60%; method B, 49%; mp >230° C.; ¹H NMR(DIMETHYLSULFOXIDE-d₆, 400 MHz) 11.89 (s, 1H, NH), 10.51 (s, 1H, NH),8.25 (d, 1H, J=1.4 Hz, C4′-H), 8.04 (d, 1H, J=7.8 Hz, C5-H), 7.85 (d,1H, J=7.7 Hz, C4″-H), 7.79 (s, 1H, C3″-H), 7.75 (d, 1H, J=8.3 Hz,C7″-H), 7.66 (dd, 1H, J=1.8, 8.9 Hz, C6′-H), 7.63 (t, 1H, J=7.4 Hz,C7-H), 7.53 (t, 1H, J=8.3 Hz, C6″-H), 7.50 (d, 1H, J=8.7 Hz, C7′-H),7.46 (t, 1H, J=7.2 Hz, C6-H), 7.39 (t, 1H, J=7.4 Hz, C5″-H), 7.31 (s,1H, C3′-H), 7.28 (d, 1H, J=7.8 Hz, C8-H), 7.00 (s, 1H, C3-H), 4.66 (dd,1H, J=5.0, 10.3 Hz, C1-H), 4.53 (d, 1H, J=10.3 Hz, C1-H), 3.28-3.32 (m,1H, C9a-H), 1.79 (dd, 1H, J=4.1, 7.6 Hz, C9-H), 1.73 (apparent t, 1H,J=4.4 Hz, C9-H); ¹³C NMR (tetrahydrofuran-d₈, 100 MHz) 185.2 (C), 162.3(C), 160.7 (C), 157.0 (C), 155.9 (C), 151.1 (C), 141.4 (C), 135.2 (C),134.1 (C), 132.8 (C), 132.2 (CH), 131.6 (C), 128.7 (C), 127.5 (CH),127.1 (CH), 126.9 (CH), 124.5 (CH), 123.4 (CH), 122.5 (CH), 120.5 (CH),114.1 (CH), 112.7 (CH), 112.4 (two CH), 110.9 (CH), 108.4 (C), 107.9(CH), 55.3 (CH₂), 33.2 (C), 29.9 (CH) 28.5 (CH₂): IR (film)_(max) 3299,1654, 1595, 1517, 1388, 1262, 1127, 744 cm⁻¹; FABHRMS (NBA) m/e 500.1610(M⁺+H, C₃₁H₂₁N₃O₄ requires 500.1610). Natural (+)-84: [α]³+91 (c 0.13,tetrahydrofuran).

N²-[(5′-(((Benzo[b]thieno-2-″-yl)carbonyl)amino)-1H-indol-2′-yl)carbonyl]-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one(86): Method A, 50%; method B, 46%; mp >230° C.; ¹H NMR(DIMETHYLSULFOXIDE-d₆, 400 MHz) 11.88 (s, 1H, NH), 10.50 (s, 1H, NH),8.36 (s, 1H, C3″-H), 8.18 (s, 1H, C4′-H), 8.06 (d, 1H, J=6.7 Hz, C7″-H),8.01 (d, 2H, J=7.2 Hz, C4″-H, C5-H), 7.61 (t, 1H, J=8.2 Hz, C7-H), 7.59(d, 1H, J=8.9 Hz, C6′-H), 7.42-7.51 (m, 4H, C6-H, C6″-H, C5″-H, C7′-H),7.28 (s, 1H, C3′-H), 7.26 (d, 1H, J=7.8 Hz, C8-H), 6.98 (s, 1H, C3-H),4.65 (dd, 1H, J=5.0, 10.3 Hz, C1-H), 4.51 (d, 1H, J=10.2 Hz, C1-H), 3.28(m, 1H, partially obscured by H₂O, C9a-H), 1.76 (dd, 1H, J=4.2, 7.6 Hz,C9-H), 1.71 (apparent t, 1H, J=4.8 Hz, C9-H); IR (film)_(max) 3321,1652, 1593, 1554, 1516, 1386, 1256, 1121 cm⁻¹; FABHRMS (NBA) m/e516.1391 (M⁺+H, C₃₁H₂₁N₃P₃S requires 516.1382). Natural (+)-86: +73 (c0.05, dimethylformamide).

N²-[(5′-(((1H-Indol-2″-yl)carbonyl)amino)benzofuran-2′-yl)carbonyl]-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one(88): Method A, 63%; method B, 45%; mp >230° C.; ¹H NMR(DIMETHYLSULFOXIDE-d₆, 400 MHz) 11.77 (s, 1H, NH), 10.38 (s, 1H, NH),8.35 (d, 1H, J=2.0 Hz, C4′-H), 8.01 (d, 1H, J=7.8 Hz, C5-H), 7.87 (s,1H, C3′-H), 7.85 (dd, 1H, J=2.1, 9.0 Hz, C6′-H), 7.75 (d, 1H, J=9.0 Hz,C7′-H), 7.68 (d, 1H, J=8.1 Hz, C4″-H), 7.61 (t, 1H, J=7.8 Hz, C7-H),7.47 (d, 1H, J=8.8 Hz, C7″-H), 7.44 (s, 1H, C3″-H), 7.44 (t, 1H, J=8.0Hz, C6-H), 7.25 (d, 1H, J=7.0 Hz, C8-H), 7.22 (t, 1H, J=8.2 Hz, C6″-H),7.07 (t, 1H, J=7.0 Hz, C5″-H), 6.91 (s, 1H, C3-H), 4.53-4.59 (m, 2H,C1-H₂), 3.27-3.28 (m, 1H, partially obscured by H₂O, C9a-H), 1.72-1.78(m, 2H, C9-H); IR (film)_(max) 3330, 1660, 1548, 1382, 1300, 1242, 1035cm⁻¹; FABHRMS (NBA) m/e 500.1600 (M⁺+H, C₃₁H₂₁N₃O₄ requires 500.1610).Natural (+)-88: +176 (c 0.09, dimethylformamide).

N²-[(5′-(((Benzofuro-2″-yl)carbonyl)amino)benzofuro-2′-yl)carbonyl]-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one(90): Method A, 93%; method B, 49%; mp >230° C.; ¹H NMR(DIMETHYLSULFOXIDE-d₆, 400 MHz) 10.74 (s, 1H, NH), 8.39 (d, 1H, J=2.0Hz, C4′-H), 8.04 (d, 1H, J=7.9 Hz, C5-H), 7.90 (s, 1H, C3′-H), 7.89 (dd,1H, J=2.2, 9.0 Hz, C6′-H), 7.87 (d, 1H, J=7.8 Hz, C4″-H), 7.82 (s, 1H,C3″-H), 7.78 (d, 1H, J=9.0 Hz, C7′-H), 7.77 (d, 1H, J=8.4 Hz, C7″-H),7.64 (t, 1H, J=7.5 Hz, C7-H), 7.54 (t, 1H, J=8.3 Hz, C6″-H), 7.47 (t,1H, J=7.6 Hz, C6-H), 7.41 (t, 1H, J=8.0 Hz, C5″-H), 7.27 (d, 1H, J=7.6Hz, C8-H), 6.94 (s, 1H, C3-H), 4.62 (dd, 1H, J=4.7, 10.5 Hz, C1-H), 4.57(d, 1H, J=10.4 Hz, C1-H), 3.30 (m, 1, partially obscured by H₂O, C9a-H),1.80 (dd, 1H, J=4.1, 7.7 Hz, C9-H), 1.76 (t, 1H, J=4.6 Hz, C9-H); IR(film)_(max) 3369, 2921, 1660, 1600, 1549, 1378, 1295, 1244, 1050 cm⁻¹;FABHRMS (NBA) m/e 501.1470 (M⁺+H, C₃₁H₂₀N₂O₅ requires 501.1450). Natural(+)-90: [α]³+90 (c 0.10, dimethylformamide).

N²-[(5′-(((Benzo[b]thieno-2″-yl)carbonyl)amino)benzofuro-2′-yl)carbonyl]-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one(92): Method B, 50%; mp >230° C.; ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz)10.72 (s, 1H, NH), 8.41 (s, 1H, C3″-H), 8.34 (d, 1H, J=2.2 Hz, C4′-H),8.09 (d, 1H, J=7.0 Hz, C5-H), 8.05 (d, 1H, J=7.0 Hz, C7″-H), 8.04 (d,1H, J=6.2 Hz, C4″-H), 7.90 (s, 1H, C3′-H), 7.85 (dd, 1H, J=2.0, 8.9 Hz,C6′-H), 7.78 (d, 1H, J=9.0 Hz, C7′-H), 7.64 (t, 1H, J=6.2 Hz, C7-H),7.45-7.55 (m, 3H, C6-H, C6″-H, C5″-H), 7.27 (d, 1H, J=8.0 Hz, C8-H),6.94 (s, 1H, C3-H), 4.58-4.62 (m, 2H, C1-H₂), 3.27-3.28 (m, 1H obscuredby H₂O, C9a-H), 1.76-1.80 (m, 2H, C9-H₂); IR (film)_(max) 2920, 2851,1661, 1599, 1555, 1466, 1381, 1297, 1243 cm⁻¹; FABHRMS (NBA) m/e517.1233 (M⁺+H, C₃₁H₂₀N₂O₄S requires 517.1222). Natural (+)-92: [α]³+69(c 0.04, dimethylformamide).

Preparation of2,4-DinitrophenylN¹-[N²-(tert-Butyloxycarbonyl)-hydrazino]-carboxylate(96) (Illustrated in FIG. 16) A suspension ofbis(2,4-dinitrophenyl)carbonate (94, 394 mg, 1.0 mmol) as prepared byGray et. al. Tetrahedron, 1977,33, 739, in 1.5 mL of Ethylacetate at 24°C. under N₂ was treated with a solution of tert-butylcarbazate fromAldrich company (132 mg, 1.0 mmol) in Ethylacetate (6 mL), and thereaction mixture was stirred for 2 h (24° C.). The reaction mixture wasfiltered through a glass filter. The filtrate was concentrated to 2 mLbelow 24° C. in vacuo and mixed with hexane (10 mL). The resultingprecipitate was collected by filtration to afford 96 (271 mg, 72% pureas a mixture with 2,4-dinitrophenol) and a second crop of crystals wasobtained from the mother liquor to afford pure 96 (12 mg) as colorlessflakes: mp 105-107° C. (hexane, colorless flakes); ¹H NMR (CDCl₃ 200MHz) 8.93 (d, 1H, J=2.7 Hz, C3-H), 8.51 (dd, 1H, J=2.7, 9.0 Hz, C5-H),7.62 (d, 1H, J=9.0 Hz, C6-H), 7.05 (br s, 1H, NH), 6.43 (br s, 1H, NH),1.48 (s, 9H, C(CH₃)₃); IR (KBr)_(max) 3414, 3268, 3112, 2978, 1754,1738, 1612, 1538, 1484, 1394, 1346, 1240, 1166, 1070, 1024, 918, 858,834, 752, 728, 642 cm⁻¹.

Preparation of3-[N¹-[N²-(tert-Butyloxycarbonyl)hydrazino]carbonyl]-1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz[e]indole(98) (Illustrated in FIG. 16). Phenol 14 (6.0 mg, 18 μmol) was treatedwith anhydrous 3N HCl-Ethylacetate (0.5 mL) at 24° C. for 20 min. thesolvent was removed in vacuo to afford crude, unstable 16quantitatively. A solution of 16 in tetrahydrofuran (0.4 mL) at 24° C.under Ar was treated sequentially with 96 (11 mg, 72% pure, 23.4 μmol,1.3 equiv) and Triethylamine (2.5 μL, 18 μmol, 1 equiv), and thereaction mixture was stirred for 5.5 h (24° C.). Flash chromatography(1.5×15 cm SiO₂, 66% Ethylacetate-hexane) afforded 98 (6.4 mg, 7.0 mgtheoretical, 91%) as a white solid: mp 221° C.; ¹H NMR(CDCl₃-dimethylformamide-d₇, 200 MHz) 9.82 (s, 1H, OH), 8.25 (d, 1H, J=8Hz, C6-H), 7.82 (s, 1H, C4-H), 7.64 (d, 1H, J=2 Hz, NH), 7.58 (d, 1H,J=8 Hz, C9-H), 7.46 (ddd, 1H, J=1.4, 7, 8 Hz, C8-H), 7.30 (ddd, 1H,J=1.4, 7, 8 Hz, C7-H), 6.86 (br s, 1H, NH), 4.24 (dd, 1H, J=3, 10 Hz,C2-H), 4.17 (t, 1H, J=10 Hz, C2-H), 3.98 (m, 1H, C-1H), 3.92 (dd, 1,J=3, 11 Hz, CHHCl), 3.37 (t, 1H, J=11 Hz, CHHCl), 1.50 (s, 9H, C(CH₃)₃);IR (KBr)_(max) 3408, 2926, 1718, 1654, 1584, 1522, 1476, 1.394, 1246,1160, 862, 758 cm⁻¹; UV (tetrahydrofuran)_(max) 318 (=9500), 308 (8200),260 (31000), 254 nm (32000); FABHRMS (DTT-DTE) m/e 392.1364 (M⁺+H,C₁₉H₂₂ClN₃O₄ requires 392.1377).

Preparation of1-(Chloromethyl)-3-(hydrazino)carbonyl-5-hydroxy-1,2-dihydro-3H-benz[e]indolehydrochloride (100) (Illustrated in FIG. 16). A sample of 98 (1.0 mg,2.6 μmol) was treated with anhydrous 3N HCl-Ethylacetate (1 mL) at 24°C. for 30 min. The solvent was removed in vacuo to afford 100 (0.9 mg,0.87 mg theoretical, 100%) as a white solid: mp 225° C. (dec); ¹H NMR(CDCl₃-DIMETHYLSULFOXIDE-d₆, 300 MHz) 10.00 (bs s, N⁺H₃), 9.96 (s, 1H,OH), 9.77 (s, 1H, CONH), 8.18 (d, 1H, J=8.3 Hz, C6-H), 7.78 (s, 1H,C4-H), 7.63 (d, 1H, J=8.3 Hz, C9-H), 7.48 (t, 1H, J=7.4 Hz, C8-H), 7.30(t, 1H, J=7.5 Hz, C7-H), 4.26 (m, 2H, C2-H), 4.07 (m, 1H, C1-H), 3.93(dd, 1H, J=2, 11 Hz, CHHCl), 3.51 (t, 1H, J=10.2 Hz, CHHCl); IR(KBr)_(max) 3400 (br), 3200 (br), 2926, 1670, 1632, 1584, 1520, 1478,1420, 1394, 1352, 1242, 1154, 1126, 1074, 1024, 756 cm⁻¹; UV(dimethylformamide)_(max) 322 (=9300), 310 (sh, 7900), 270 nm (23000);FABHRMS (DTT-DTE) m/e 292.0867 (M⁺+H, C₁₄H₁₄ClN₃O₂ requires 292.0853).

Preparation of compounds 102 and 104 (Illustrated in FIG. 16). Asolution of 98 or 100 (1.0 equiv) in tetrahydrofuran (0.02 molar) wastreated with 0.02 molar of 5% aqueous NaHCO₃ and the two-phase mixturewas stirred at 24° C. for 5 h under N₂. The reaction mixture wasextracted with Ethylacetate (3×). The organic layer was dried (Na₂SO₄)and concentrated. Flash chromatography affords 102 and 104 as a paleyellow solid.

Preparation of N¹-[N²-(tert-Butyloxycarbonyl)hydrazino]carbonyl-CDPI₂(108) (Illustrated in FIG. 17). N-BOC-CDPI₂ (106, 6.2 mg, 12.8 μmol) aspreviously described in Boger et. al J. Org. Chem. 1987, 52, 1521, wastreated with CF₃CO₂H (0.5 mL) at 24° C. for 1 h. The CF₃CO₂H was removedby a stream of N₂ and the residue was dried in vacuo. A solution of thecrude salt in dimethylformamide (0.2 mL) at 24° C. under Ar was treatedsequentially with 96 (72% pure in 2,4 -dinitrophenol, 9.1 mg, 19.3 μmol,1.5 equiv) and Triethylamine (1.8 μL, 12.8 μmol, 1 equiv) and thereaction mixture was stirred for 19 h (24° C.). The solvent was removedin vacuo and the residue was washed with saturated aqueous NaHCO₃ (1mL), H₂O (0.5 mL), 10% aqueous citric acid (1 mL), and H₂O (4×1 mL).Drying the solid in vacuo afforded 108 (6.3 mg, 6.9 mg theoretical, 91%)as a pale yellow solid: mp 257° C. (dec); ¹H NMR (DIMETHYLSULFOXIDE-d₆,300 MHz) 11.84 (s, 1H, NH), 11.61 (s, 1H, NH), 8.62 (s, 1H, CONH), 8.42(s, 1H, CONH), 8.28 (br d, 1H, J=9 Hz, C4-H), 7.94 (d, 1H, J=8.9 Hz,C4′-H), 7.32 (d, 1H, J=9 Hz, C5-H), 7.26 (d, 1H, J=8.9 Hz, C5′-H), 7.06(s, 1H, C8′-H), 6.98 (s, 1H, C8-H), 4.64 (t, 2H, J=8.3 Hz, C2-H₂), 4.02(t, 2H, J=8.3 Hz, C2′-H), 3.2-3.6 (m, 4H, partly obscured by H₂O, C1-H₂,C1′-H₂), 1.43 (s, 9H, C(CH₃)₃); IR (KBr)_(max) 3424, 1686, 1508, 1438,1372, 1160, 800, 684 cm⁻¹.

Preparation ofN¹[N²-(tert-Butyloxycarbonyl)hydrazino]carbonyl-seco-CBI-CDPI₂ (110)(Illustrated in FIG. 17). A slurry of crude 16 freshly prepared from 14(3.7 mg, 11. 1 μmol), 1-(3-DIMETHYLAMINOPROPYL)-3-ETHYLCARBODIIMIDEHYDROCHLORIDE (EDCI) (6.4 mg, 33 μmol, 3 equiv), and 108 (6.0 mg, 11.1μmol, 1 equiv) in dimethylformamide (0.2 mL) at 24° C. under Ar wasvigorously stirred for 10 h. The solvent was removed in vacuo and theresidue was washed with H₂O (2×2 mL) and dried in vacuo. Flashchromatography (0.5×5 cm SiO₂, 0-66% dimethylformamide-toluene gradientelution) afforded 110 (5.5 mg, 8.4 mg theoretical, 65%) as a pale yellowsolid: mp 250° C. (dec); ¹H NMR (DIMETHYLSULFOXIDE-d₆, 300 MHz) 11.83(s, 1H, NH), 11.63 (s, 1H, NH), 10.45 (s, 1H, OH), 8.63 (s, 1H, CONH),8.43 (s, 1H, CONH), 8.29 (br d, 1H, J=9 Hz, C4′-H), 8.13 (d, 1H, J=8.5Hz, C6-H), 7.99 (s, 1H, C4-H), 7.95 (d, 1H, J=8.9 Hz, C4″-H), 7.87 (d,1H, J=8.3 Hz, C9-H), 7.54 (t, 1H, J=7.6 Hz, C8-H), 7.40 (d, 1H, J=9.3Hz, C5′-H), 7.38 (t, 1H, J=7.7 Hz, C7-H), 7.27 (d, 1H, J=8.9 Hz, C5″-H),7.19 (s, 1H, C8′-H), 7.01 (s, 1H, C8″-H), 4.85 (t, 1H, J=10 Hz, C2-H),4.68 (t, 2H, J=8 Hz, C2′-H₂), 4.59 (d, 1H, J=10 Hz, C2-H), 4.26 (m, 1H,CHHCl), 4.03 (t, 2H, J=8 Hz, C2″-H), 3.9-4.0 (m, 2H, C1-H), CHHCl),3.2-3.6 (m, 4H, partly obscured by H₂O, Cl′-H₂, Cl″-H₂), 1.44 (s, 9H,C(CH₃)₃); IR (KBr)_(max) 3410, 3315, 2962, 2927, 1664, 1610, 1582, 1508,1416, 1370, 1340, 1262, 1158, 1098, 802, 762, 528 cm⁻¹; UV(dimethylformamide)_(max) 340 (=43000), 310 (44000), 270 nm (26000);FABHRMS (DTT-DTE) m/e 760.2635 (M⁺+H, C₄₁H₃₈ClN₇O₆ requires 760.2650).

Preparation of compound 112 (Illustrated in FIG. 17) A sample of 110(1.0 equiv.) is treated with anhydrous 3N HCl-Ethylacetate (0.25 M) at24° C. for 30 min. The solvent is then removed in vacuo to afford 112 asa white solid.

Preparation of 2-[(Benzyloxy)methyl]pyrrolo[3,2-e]benzoxazole (116)(Illustrated in FIG. 25). A solution of 5-hydroxyindole 114 (499 mg,3.75 mmol) from Aldrich company and 2-(benzyloxy)ethylamine (1.13 g,7.50 mmol, 2 equiv) monobenzylated from Aldrich company in anhydrousethylene glycol dimethyl ether (DME, 120 mL) was cooled to 0° C. andactivated MnO₂ (15 g, 30 wt equiv) was added. The reaction mixture wasallowed to stir at 24° C. for 14 h before filtration through a Celitepad to remove MnO₂. The solvent was removed in vacuo. Flashchromatography (SiO₂, 2.5×25 cm, 40-50% Ethylacetate-hexane gradientelution) afforded 116 (500 mg, 1.04 g theoretical, 48%) as a paleorange-yellow oil: ¹H NMR (CDCl₃, 400 MHz) 8.98 (br s, 1H, NH),7.27-7.40 (m, 8H, ArH), 6.92-6.93 (m, 1H, ArH), 4.84 (s, 2H, PhCH₂),4.70 (s, 2H, C2-CH₂); ¹³C NMR (CDCl₃, 100 MHz) 161.4 (C), 146.1 (C),137.1 (C), 133.7 (C), 128.5 (two CH), 128.1 (two CH), 128.05 (CH), 127.8(C), 125.3 (CH), 119.4 (C), 109.3 (CH), 104.8 (CH), 100.1 (CH), 73.2(CH₂), 64.6 (CH₂); IR (neat)_(max) 3265, 2862, 1671, 1452, 1367, 1212,1089, 738, 698 cm⁻¹; FABHRMS (NBA) m/e 279.1140 (M⁺+H, C₁₇H₁₄N₂P₂requires 279.1134).

Preparation of 2-(Hydroxymethyl)pyrrolo[3,2-e]benzoxazole (118)(Illustrated in FIG. 25). A solution of 116 (67 mg, 0.24 mmol) inEthanol (4 mL) was treated with 3 drops of conc HCl followed by 10% Pd—C(34 mg, 0.5 wt equiv). The reaction mixture was stirred at 24° C. under1 atm of H₂ for 30 min, and neutralized with the addition ofTriethylamine. The mixture was filtered through a Celite pad to removethe catalyst and the solvent was removed in vacuo. Flash chromatography(SiO₂, 1.0×20 cm, 60-80% Ethylacetate-hexane gradient elution) afforded118 (31.5 mg, 45.1 mg theoretical, 70%) as a white crystalline solid: mp169-171.5° C. (CH₃OH—CH₂Cl₂); ¹H NMR (CD₃OD, 400 MHz) 7.42 (dd, 1H,J=0.8, 8.8 Hz, ArH), 7.36 (d, 1H, J=3.1 Hz, C7-H), 7.32 (d, 1H, J=8.8Hz, ArH), 6.78 (dd, 1H, J=0.8, 3.1 Hz, C8-H), 4.82 (s, 2H, CH₂OH); ¹³CNMR (CD₃OD, 100 MHz) 165.5 (C), 147.0 (C), 135.6 (C), 133.3 (C), 126.8(CH), 120.3 (C), 110.6 (CH), 105.0 (CH), 99.7 (CH), 58.2 (CH₂); IR(film)_(max) 3266, 1566, 1438, 1364, 1221, 1083, 1037, 775, 735, 668cm⁻¹; FABHRMS (NBA) m/e 189.0668 (M⁺+H, C₁₀H₈N₂O₂ requires 189.0664).Anal. Calcd for C₁₀H₈N₂O₂: C, 63.81; H, 4.29; N, 14.89. Found: C, 63.50;H, 4.20; N, 14.50.

Preparation of methyl Pyrrolo[3,2-e]benzoxazole-2-carboxylate (120)(Illustrated in FIG. 25). A solution containing NaCN (42 mg, 0.85 mmol,5 equiv) and activated MnO₂ (148 mg, 1.7 mmol, 10 equiv) in 10.5 mL ofCH₃OH was treated with a solution of 118 (32 mg, 0.17 mmol) in CH₃OH(5.5 mL) at 0° C. under Ar. The reaction mixture was allowed to warm to24° C. and was stirred for 4 h. The reaction mixture was filteredthrough a Celite pad (2×30 mL Ethylacetate wash) to remove MnO₂ and thecombined organic layer was washed with H₂O, saturated aqueous NaCl,dried (Na₂SO₄), and concentrated in vacuo. Flash chromatography (SiO₂,1×15 cm, 40% Ethylacetate-hexane) afforded 120 (37 mg, 37 mgtheoretical, 100%) as an off-white solid: mp 207-208° C.(Ethylacetate-hexane); ¹H NMR (CDCl₃, 400 MHz) 8.69 (br s, 1H, NH), 7.57(d, 1H, J=8.9 Hz, ArH), 7.46 (d, 1H, J=8.9 Hz, ArH), 7.39 (t, 1H, J=2.8Hz, C7-H), 7.05-7.06 (m, 1H, C-8H), 4.09 (s, 3H, CH₃); ¹³C NMR (CDCl₃,100 MHz) 157.2 (C), 151.1 (C), 146.7 (C), 133.9 (C), 133.2 (C), 125.8(CH), 119.8 (C), 113.0 (CH), 104.7 (CH), 100.4 (CH), 53.5 (CH₃); IR(film)_(max) 3356, 2921, 1738, 1537, 1437, 1371, 1148 cm⁻¹; FABHRMS(NBA) m/e 217.0610 (M⁺+H, C₁₁H₈N₂O₃ requires 217.0613). Anal. Calcd forC₁₁H₈N₂O₃: C, 61.10; H, 3.73; N, 12.96. Found: C, 60.92; H, 3.71; N,12.79.

Preparation of Methyl1,2-Dihydro-3H-pyrrolo[3,2-e]benzoxazole-7-carboxylate (122)(Illustrated in FIG. 25). Compound 120 (47.6 mg, 0.22 mmol) wasdissolved in CF₃CO₂H (1 mL) and cooled to 0° C. The mixture was stirredfor 10 min before Et₃SiH (355 μL, 2.20 mmol, 10 equiv) was added to thereaction mixture. The mixture was warmed to 24° C. and stirred for 4.5h. The solvent was removed under a stream of N₂ and the residue wasdissolved in CH₂Cl₂ (10 mL), and washed with saturated aqueous NaHCO₃(10 mL). The organic layer was dried (Na₂SO₄) and concentrated in vacuoto afford crude 9 as a yellow solid which was used directly in the nextstep without further purification due to its propensity to air oxidizeback to starting material. For crude 122: ¹H NMR (CDCl₃, 400 MHz) 7.25(d, 1H, J=8.7 Hz, C5-H), 6.80 (d, 1H, J=8.7 Hz, C4-H), 4.01 (s, 3H,CH₃), 3.65 (t, 2H, J=8.6 Hz, C2-H₂), 3.31 (t, 2H, J=8.6 Hz, C1-H₂);FABHRMS (NBA) m/e 219.0768 (M⁺+H, C₁₁H₁₀N₂O₃ requires 219.0770).

Preparation of Methyl3-Carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]benzoxazole-7-carboxylate (124)(Illustrated in FIG. 25). A solution of crude 122 (from 0.22 mmol of120) in anhydrous CH₂Cl₂ (2 mL) was treated with 85% trimethylsilylisocyanate (Me₃SiNCO, 174 μL, 1.10 mmol, 5 equiv) and the mixture wasstirred at 24° C. under N₂ for 4 h. The resulting insoluble residue wascollected by centrifugation and washed with CH₂Cl₂ (2×3 mL) and CH₃OH (3mL). Drying the solid in vacuo afforded pure 124 (32.7 mg, 57.4 mgtheoretical, 57% from 120) as a pale yellow solid: mp >230° C. (dec); ¹HNMR (DIMETHYLSULFOXIDE-d₆, 400 MHz) 8.20 (d, 1H, J=9.0 Hz, C4-H), 7.57(d, 1H, J=9.0 Hz, C5-H), 6.35 (br s, 2H, NH₂), 4.04 (t, 2H, J=8.9 Hz,C2-H₂), 3.96 (s, 3H, CH₃), 3.38 (t, 2H, J=8.9 Hz, C1-H₂); ¹³C NMR(DIMETHYLSULFOXIDE-d₆, 100 MHz) 156.5 (C), 155.9 (two C), 146.1 (C),143.0 (C), 136.9 (C), 122.0 (C), 115.2 (CH), 109.3 (CH), 53.4 (CH₃),48.2 (CH₂), 25.2 (CH₂); IR (film)_(max) 3448, 3179, 1727, 1675, 1606,1543, 1487, 1423, 1321, 1229, 1155, 1140, 1028, 818 cm⁻¹; FABHRMS (NBA)m/e 262.0830 (M⁺+H, C₁₂H₁₁N₃O₄ requires 262.0828). Anal. Calcd forC₁₂H₁₁N₃O₄: C, 55.16; H, 4.25; N, 16.09. Found: C, 54.93; H, 4.17; N,15.95.

Preparation of Methyl 3-(tert-Butyloxycarbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]benzoxazole-7-carboxylate (126) (Illustrated in FIG. 25). Asolution of crude 122 (1.8 mg, 0.008 mmol) dissolved in tetrahydrofuran(100 μL) was treated with di-tert-butyl dicarbonate (3.6 mg, 3.8 μL,0.016 mmol, 2 equiv). The reaction mixture was stirred at 24° C. for 2 hand 4° C. for 24 h. The solvent was removed in vacuo and flashchromatography (SiO₂, 20-40% Ethylacetate-hexane gradient elution)afforded 126 (2.0 mg, 2.6 mg theoretical, 76%) as a pale yellow solid:¹H NMR (CDCl₃, 400 MHz) 8.17 (br s, 1H, C4-H), 7.43 (d, 1H, J=9.0 Hz,C5-H), 4.13 (t, 2H, J=8.8 Hz, C2-H₂), 4.07 (s, 3H, CH₃), 3.41 (t, 2H,J=8.8 Hz, C1-H₂), 1.56 (s, 9H, C(CH₃)₃); FABHRMS (NBA) m/e 319.1290(M⁺+H, C₁₆H₁₈N₂O₅ requires 319.1294).

Preparation or3-Carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]benzoxazole-7-carboxylic Acid(128) (Illustrated in FIG. 25). A suspension of 124 (27 mg, 0.103 mmol)and LiOH H₂O (8.8 mg, 0.21 mmol, 2 equiv) in tetrahydrofuran-CH₃OH—H₂O(3:1:1, 2.5 mL) was stirred at 24° C. for 4 h. The solvent was removedunder a stream of N₂ and the residual solid was suspended in H₂O (2 mL)and acidified with 1N aqueous HCl to pH 1. The insoluble residue wascollected by centrifugation and washed with H₂O (2×3 mL). Drying thesolid in vacuo afforded 128 (25 mg, 25 mg theoretical, 100%) as a paleyellow powder: mp >230° C. (dec); ¹H NMR (CF₃CO₂D, 400 MHz) 8.31 (d, 1H,J=9.4 Hz, C4-H), 7.76 (d, 1H, J=9.4 Hz, C5-H), 4.41 (t, 1H, J=8.4 Hz,C2-H₂), 3.73 (t, 1H, J=8.4 Hz, C1-H₂); ¹³C NMR (DIMETHYLSULFOXIDE-d₆,100 MHz) 155.9 (C), 154.8 (two C), 145.2 (C), 142.0 (C), 136.3 (C),120.9 (C), 112.2 (CH), 108.4 (CH), 48.1 (CH₂), 25.2 (CH₂); IR(film)_(max) 3476, 3174, 1677, 1606, 1481, 1419, 1369, 1234, 1061, 815cm⁻¹; FABHRMS (NBA) m/e 248.0674 (M⁺+H, C₁₁H₉N₃O, requires 248.0671).

Preparation of 1-(tert-Butyloxycarbonyl)-5-nitroindole (132)(Illustrated in FIG. 26).

A solution of 5-nitroindole (130, 2.0 g, 12.3 mmol) from Aldrich companyand 4-DIMETHYLAMINOPYRIDINE (226 mg, 1.85 mmol, 0.15 equiv) in dioxane(90 mL) was treated with di-tert-butyl dicarbonate (5.38 g, 24.7 mmol, 2equiv), and the reaction mixture was stirred at 24° C. for 10-15 minbefore the solvent was removed in vacuo. Flash chromatography (SiO₂,2.5×25 cm, 20-50% Ethylacetate-hexane gradient elution) afforded 132(3.17 g, 3.17 g theoretical, 100%) as an off-white solid: mp 135-137° C.(CH₂Cl₂, off-white fine needles); ¹H NMR (CDCl₃, 400 MHz) 8.42 (d, 1H,J=2.1 Hz, C4-H), 8.21 (d, 1H, J=9.1 Hz, C7-H), 8.14 (dd, 1H, J=2.1, 9.1Hz, C6-H), 7.70 (d, 1H, J=3.8 Hz, C2-H), 6.67 (d, 1H, J=3.8 Hz, C3-H),1.67 (s, 9, C(CH₃)₃); ¹³C NMR (CDCl₃, 100 MHz) 148.9 (C), 143.6 (C),138.2 (C), 130.2 (C), 128.8 (CH), 119.4 (CH), 117.2 (CH), 115.2 (CH),107.8 (CH), 85.1 (C), 28.0 (three CH₃); IR (film)_(max) 2982, 1737,1515, 1462, 1329, 1285, 1254, 1156, 1027, 904, 768, 745 cm⁻¹; FABHRMS(NBA) m/e 263.1043 (M⁺+H, C₁₃H₁₄N₂O₄ requires 263.1032). Anal. Calcd forC₁₃H₁₄N₂O,: C, 59.52; H, 5.38; N, 10.69. Found: C, 59.53; H 5.36; N,10.53.

Preparation of 5-Amino-1-(tert-butyloxycarbonyl)indole (134)(Illustrated in FIG. 26). A solution of 132 (1.0 g, 3.81 mmol) inEthylacetate (30 mL) was treated with 10% Pd—C (500 mg, 0.5 wt equiv)and the mixture was stirred under 1 atm of H₂ at 24° C. for 5 h. Thecatalyst was removed by filtration through Celite, and the solvent wasremoved in vacuo. Flash chromatography (SiO₂, 2×20 cm, 40-60%Ethylacetate-hexane gradient elution) afforded 134 (593 mg, 884 mgtheoretical, 67%) as a pale brown oil: ¹H NMR (CDCl₃, 400 MHz) 7.90 (brs, 1H, C7-H), 7.50 (br s, 1H, C2-H), 6.82 (d, 1H, J=2.3 Hz, C4-H), 6.70(dd, 1H, J=2.3, 8.7 Hz, C6-H) 6.39 (d, 1H, J=3.6 Hz, C3-H), 3.43 (br s,2H, NH₂), 1.65 (s, 9H, C(CH₃)₃); ¹³C NMR (CDCl₃, 100 MHz) 149.8 (C),142.3 (C), 131.7 (C), 129.1 (C), 126.2 (CH), 115.7 (CH), 113.8 (CH),106.9 (CH), 106.0 (CH), 83.2 (C), 28.3 (three CH₃); IR (neat)_(max)3359, 1725, 1477, 1454, 1380, 1357, 1343, 1285, 1229, 1166, 1150, 1132,1024 cm⁻¹; FABHRMS (NBA) m/e 232.1212 (M⁺, C₁₃H₁₆N₂O₂ requires232.1212).

Preparation of5-(2-Benzyloxyacetyl)amino-1-(tert-butyloxy-carbonyl)-indole (136)(Illustrated in FIG. 26). A solution of 134 (991 mg, 4.27 mmol) andK₂CO₃ (400 mg, 5.12 mmol, 1.2 equiv) in tetrahydrofuran (75 mL) wascooled to 0° C. and stirred for 10 min before benzyloxyacetyl chloride(851 μL, 5.12 mmol, 1.2 equiv) was added. The reaction mixture then wasallowed to warm to 24° C. and stirred under N₂ for 2 h. The mixture wasdiluted with H₂O (100 mL), extracted with Ethylacetate (3×150 mL), dried(Na₂SO₄) and concentrated. Flash chromatography (SiO₂, 2×20 cm, 40-50%Ethylacetate-hexane gradient elution) afforded 136 (1.53 g, 1.62 gtheoretical, 94%) as a pale yellow oil: ¹H NMR (CDCl₃, 400 MHz) 8.37 (brs, 1H, NH), 8.06 (br d, 1H, J=8.6 Hz, C7-H), 7.95 (d, 1H, J=2.0 Hz,C4-H), 7.56 (d, 1H, J=3.9 Hz, C2-H), 7.34-7.41 (m, 5H, C₆H₅), 7.27 (dd,1H, J=2.0, 8.6 Hz, C6-H), 6.51 (d, 1H, J=3.9 Hz, C3-H), 4.65 (s, 2H,PhCH₂), 4.11 (s, 2H, COCH₂), 1.65 (s, 9H, C(CH₃)₃); ¹³C NMR (CDCl₃, 62.5MHz) 167.4 (C), 149.5 (C), 136.5 (C), 132.2 (two C), 130.9 (C), 128.7(two CH), 128.3 (CH), 128.0 (two CH), 126.6 (CH), 116.9 (CH), 115.3(CH), 112.1 (CH), 107.3 (CH), 83.6 (C), 73.7 (CH), 69.6 (CH), 28.1(three CH₃); IR (neat)_(max) 3381, 2978, 1732, 1688, 1537, 1473, 1372,1131, 745, 699 cm⁻¹; FABHRMS (NBA) m/e 381.1823 (M⁺+H, C₂₂H₂₄N₂O₄requires 381.1814).

Preparation of5-(2-Benzyloxyacetyl)amino-1-(tert-butyloxycarbonyl)-4-nitroindole (138)(Illustrated in FIG. 26). Compound 136 (783 mg, 2.06 mmol) was dissolvedin CH₃NO₂ (38 mL), cooled to 0° C., and treated with 65% HNO₃ (1.1 mL).The mixture was warmed to 24° C. and stirred for 3 h before it wasdiluted with H₂O (50 mL) and extracted with CH₂Cl₂ (3×40 mL). Theorganic layer was dried (Na₂SO₄) and concentrated. Flash chromatography(SiO₂, 2×20 cm, 20-40% Ethylacetate-hexane gradient elution) afforded138 (570 mg, 878 mg theoretical, 65%) as a bright yellow solid: mp145-146.5° C. (CH₂Cl₂, light yellow flakes); ¹H NMR (CDCl₃, 400 MHz)11.40 (br s, 1H, NH), 8.63 (d, 1H, J=9.4 Hz, ArH), 8.40 (d, 1H, J=9.4Hz, ArH), 7.71 (d, 1H, J=4.0 Hz, C2-H), 7.27-7.40 (m, 5H, C₆H₅), 7.17(d, 1H, J=4.0 Hz, C3-H), 4.68 (s, 2H, PhCH₂), 4.10 (s, 2H, COCH₂), 1.62(s, 9H, C(CH₃)₃); ¹³C NMR (CDCl₃, 100 MHz) 168.9 (C), 148.8 (C), 136.5(C), 132.2 (C), 130.7 (C), 129.8 (CH), 129.6 (C), 128.6 (two CH), 128.2(CH), 128.0 (two CH), 125.7 (C), 122.2 (CH), 117.9 (CH), 107.2 (CH),85.2 (C), 73.8 (CH₂), 69.7 (CH₂), 28.1 (three CH₃); IR (film)_(max)3319, 1737, 1701, 1491, 1372, 1323, 1288, 1152, 1108 cm⁻¹; FABHRMS (NBA)m/e 425.1605 (M⁺+H, C₂₂H₂₃N₃O₆ requires 425.1587). Anal. Calcd forC₂₂H₂₃N₃O₆: C, 62.09; H, 5.45; N, 9.88. Found: C, 61.84; H, 5.47; N,9.99.

Preparation of4-Amino-5-(2-benzyloxyacetyl)amino-1-(tertbutyloxy-carbonyl)-indole(140) (Illustrated in FIG. 26). Method A. Compound 138 (212 mg, 0.50mmol) was dissolved in tetrahydrofuran (3.5 mL) and treated with asolution of Na₂S₂O₄ (870 mg, 5.0 mmol, 10 equiv) in H₂O (3.5 mL). Thereaction mixture was stirred at 24° C. under N₂ for 20 h before it wasdiluted with H₂O (10 mL), and extracted with Ethylacetate (3×10 mL). Theorganic layer was dried (Na₂SO₄) and concentrated. Flash chromatography(SiO₂, 1.5×20 cm, 50-60% Ethylacetate-hexane gradient elution) afforded140 (138 mg, 197 mg theoretical, 70%) as a pale yellow oil identical inall respects to the sample described below.

Method B. A solution of 138 (910 mg, 2.13 mmol) in Ethylacetate (45 mL)was treated with 10% Pd—C (455 mg, 0.5 wt equiv) and the mixture wasstirred under 1 atm of H₂ at 24° C. for 3 h. The catalyst was removed byfiltration through Celite, and the solvent was removed in vacuo. Flashchromatography (SiO₂, 2×25 cm, 60% Ethylacetate-hexane) afforded 140(776 mg, 846 mg theoretical, 92%) as a pale yellow oil (no debenzylationproduct was detected): ¹H NMR (CDCl₃, 400 MHz) 8.23 (br s, 1H, NH), 7.55(d, 1H, J=8.7 Hz, ArH), 7.49 (d, 1H, J=3.8 Hz, C2-H), 7.34-7.39 (m, 5H,C₆H₅), 7.01 (d, 1H, J=8.7 Hz, ArH), 6.50 (d, 1H, J=3.8 Hz, C3-H), 4.67(s, 2H, PhCH₂), 4.17 (s, 2H, COCH₂), 4.16 (br s, 2H, NH₂), 1.64 (s, 9H,C(CH₃)₃); ¹³C NMR (CDCl₃, 100 MHz) 168.4 (C), 149.8 (C), 136.8 (C),134.6 (C), 134.5 (C), 128.8 (two CH), 128.4 (CH), 128.2 (two CH), 124.8(CH), 122.7 (CH), 120.6 (C), 116.1 (C), 106.2 (CH), 103.9 (CH), 83.8(C), 73.8 (CH₂), 69.7 (CH₂), 28.3 (three CH₃); IR (neat)_(max) 3362,2978, 1731, 1676, 1491, 1350, 1299, 1152, 1126 cm⁻¹; FABHRMS (NBA-CsI)m/e 528.0878 (M⁺+Cs, C₂₂H₂₅N₃O₄ requires 528.0899). Anal. Calcd forC₂₂H₂₅N₃O₄: C, 66.80; H, 6.38; N, 10.63. Found: C, 66.58; H, 6.34; N,10.39.

Preparation of2-[(Benzyloxy)methyl]-6-(tert-butyloxycarbonyl)-pyrrolo-3,2-e]benzimidazole(142) (Illustrated in FIG. 26). Compound 140 (192 mg, 0.484 mmol) wasdissolved in tetrahydrofuran (25 mL) and treated with a solution oftetrahydrofuran (5 mL) containing 2 drops of conc H₂SO₄. The mixture wasstirred at 24° C. under N₂ for 24 h before the reaction was neutralizedwith the addition of saturated aqueous NaHCO₃ (20 mL). The mixture wasextracted with Ethylacetate (3×20 mL) and the organic layer wasconcentrated in vacuo. Flash chromatography (SiO₂, 1.5×20 cm, 40-50%Ethylacetate-hexane gradient elution) afforded 142 (181 mg, 183 mgtheoretical, 99%) as a pale orange oil: ¹H NMR (CDCl₃, 400 MHz) 8.15 (d,1H, J=8.8 Hz, C5-H), 7.66(d, 1H, J=3.5 Hz, C7-H), 7.45 (br d, 1H, J=8.8Hz, C4-H), 7.32-7.36 (m, 5H, C₆H₅), 6.92 (br s, 1H, C8-H), 4.91 (s, 2H,PhCH₂), 4.65 (s, 2H, C2-CH₂), 1.69 (s, 9H, C(CH₃)₃); IR (neat)_(max)2978, 1732, 1436, 1370, 1343, 1286, 1150, 1128 cm⁻¹; FABHRMS (NBA) m/e378.1826 (M⁺+H, C₂₂H₂₃N₃O₃ requires 378.1818).

Preparation of 6-(tert-Butyloxycarbonyl)-2-(hydroxymethyl)pyrrolo[3,2-e]benzimidazole (144) (Illustrated in FIG. 26). A solution of 142 (677mg, 1.79 mmol) in Ethanol (20 mL) was treated with 3 drops of conc HClfollowed by 10% Pd—C (340 mg, 0.5 wt equiv). The reaction mixture wasstirred at 24° C. under 1 atm of H₂ for 5 h before being quenched withthe addition of several drops of Triethylamine. The catalyst was removedby filtration through Celite, and the solvent was removed in vacuo.Flash chromatography (SiO₂, 2×25 cm, 10-20% CH₃OH-Ethylacetate gradientelution) afforded 144 (481 mg, 516 mg theoretical, 93%) as an off-whitepowder: mp 152° C. (dec, CH₃OH—CH₂Cl₂); ¹H NMR (CD₃OD, 400 MHz) 8.10 (d,1H, J=9.0 Hz, C5-H), 7.67 (d, 1H, J=3.7 Hz, C7-H), 7.44 (d, 1H, J=9.0Hz, C4-H), 6.93 (d, 1H, J=3.7 Hz, C8-H), 4.87 (s, 2H, CH₂OH), 1.69 (s,9H, C(CH₃)₃); ¹³C NMR (CD₃OD-CDCl₃, 400 MHz) 154.0 (C), 150.9 (C), 133.2(C), 132.7 (C), 132.0 (C), 126.1 (CH), 119.7 (C), 111.5 (CH), 110.9(CH), 104.9 (CH), 84.6 (C), 58.6 (CH₂), 28.4 (three CH₃); IR(film)_(max) 3179, 2920, 1729, 1676, 1365, 1342, 1289, 1150, 1126 cm⁻¹;FABHRMS (NBA-NaI) m/e 310.1160 (M⁺+Na, C₁₅H₁₇N₃O₃ requires 310.1168).

Methyl6-(tert-Butyloxycarbonyl)pyrrolo[3,2-e]benzimidazole-2-carboxylate (146)(Illustrated in FIG. 26). A solution containing NaCN (478 mg, 9.75 mmol,5 equiv) and activated MnO₂ (1.69 g, 19.5 mmol, 10 equiv) in CH₃OH (42mL) was treated with a solution of 144 (560 mg, 1.95 mmol) in CH₃OH (17mL) at 0° C. under Ar. The reaction mixture was allowed to warm to 4° C.and was stirred for 8 h. The reaction mixture was filtered through aCelite pad (Ethylacetate wash) to remove MnO₂. Ethylacetate was added(150 mL total) and the combined organic layer was washed with H₂O (100mL), saturated aqueous NaCl, dried (Na₂SO₄), and concentrated in vacuo.Flash chromatography (SiO₂, 2×25 cm, 60-80% Ethylacetate-hexane gradientelution) afforded 146 (485 mg, 615 mg theoretical, 79%) as a lightyellow foam: ¹H NMR (CDCl₃, 400 MHz) 8.26 (d, 1H, J=9.0 Hz, C5-H), 7.64(d, 1H, J=3.6 Hz, C7-H), 7.49 (d, 1H, J=9.0 Hz, C4-H), 6.98 (br s, 1H,C8-H), 4.01 (s, 3H, CO₂CH₃), 1.62 (s, 9H, C(CH₃)₃); IR (film)_(max)3378, 1729, 1364, 1341, 1319, 1243, 1146, 1127 cm⁻¹; FABHRMS (NBA) m/e316.1299 (M⁺+H, C₁₆H₁₇N₃O₄ requires 316.1297). Anal. Calcd forC₁₆H₁₇N₃O₄: C, 60.93; H, 5.44; N, 13.33. Found: C, 60.84, H, 5.53; N,12.97.

Preparation of Methyl Pyrrolo[3,2-e]benzimidazole-2-carboxylate (148)(Illustrated in FIG. 26). Compound 146 (100 mg, 0.32 mmol) was treatedwith anhydrous 3M HCl in Ethylacetate (10 mL) at 24° C. for 5 h. Thereaction then was neutralized with saturated aqueous NaHCO₃ to pH 7-8and extracted with Ethylacetate (2×10 mL) and CH₃CN (2×15 mL). Thecombined organic layer was concentrated in vacuo to afford 148 as ayellow solid which was used in the next reaction without furtherpurification. For 148: mp 182° C. (dec, CH₃OH—CH₂Cl₂, light yellowpowder); ¹H NMR (CD₃OD, 400 MHz) 7.45 (d, 1H, J=8.8 Hz, ArH), 7.37 (d,1H, J=8.8 Hz, ArH), 7.29 (d, 1H, J=3.0 Hz, C7-H), 6.85 (d, 1H, J=3.0 Hz,C8-H), 4.02 (s, 3H, CO₂CH₃); IR (film)_(max) 3380, 1716, 1631, 1518,1434, 1387, 1314, 1238 cm⁻¹; FABHRMS (NBA) m/e 216.0779 (M⁺+H, C₁₁H₉N₃O₂requires 216.0773).

Preparation of Methyl3-Carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]benzimidazole-7-carboxylate(152) (Illustrated in FIG. 26). Crude 148 prepared above (0.32 mmoltheoretical) was treated with CF₃CO₂H (2.5 mL) and the mixture wasstirred at 24° C. for 40 min. The reaction mixture was cooled to 0° C.before Et₃SiH (510 μL, 3.17 mmol, 10 equiv) was added. The reactionmixture was warmed to 24° C. and stirred for 6 h. The solvent wasremoved under a stream of N₂ and the dry residue was dissolved in CH₂Cl₂(20 mL). Several drops of CH₃OH were added to help dissolve the residue.The organic solution was washed with saturated aqueous NaHCO₃ andconcentrated in vacuo to afford 150 as a bright yellow solid which wasused directly in the next reaction without further purification due toits propensity to air oxidize back to starting material. A solution of150 dissolved in 10 mL of CH₂Cl₂-dimethylformamide (10:1) was treatedwith 85% Me₃SiNCO (220 μL, 1.338 mmol, 5 equiv). The reaction mixturewas stirred at 24° C. for 8 h. The solvent was removed in vacuo, and thedry residue was slurried in CH₂Cl₂ (5 mL). The sample was collected bycentrifugation, washed with CH₂Cl₂ (2×) and CH₃OH (1×) to afford pure152 (55.4 mg, 82.5 mg theoretical, 67% from 146) as a light gray solid:mp >230° C. (dec); ¹H NMR (CF₃CO₂D, 400 MHz) 8.42 (d, 1H, J=9.4 Hz,C4-H), 7.78 (d, 1H, J=9.4 Hz, C5-H), 4.35 (t, 2H, J=8.4 Hz, C2-H₂), 4.21(s, 3H, CO₂CH₃), 3.64 (t, 2H, J=8.4 Hz, C1-H₂), a doubling of the ¹H NMRsignals was observed when the spectrum was recorded inDIMETHYLSULFOXIDE-d₆ which we attribute to the two accessible tautomericforms of 152; ¹³C NMR (CF₃CO₂D, 100 MHz) 160.8 (C), 156.6 (C), 145.7(C), 139.4 (C), 130.5 (C), 130.3 (C), 121.9 (CH), 119.8 (C), 117.0 (CH),57.5 (CH₃), 50.9 (CH₂), 27.0 (CH₂); IR (KBr)_(max) 3406, 3187, 3027,1727, 1664, 1441, 1394, 1209, 769 cm⁻¹; FABHRMS (NBA) m/e 261.0993(M⁺+H, C₁₂H₁₂N₄O₃ requires 261.0988).

Preparation of3-Carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]benzimidazole-7-carboxylic Acid(154) (Illustrated in FIG. 26). A suspension of 152 (50 mg, 0.19 mmol)in 6 mL of tetrahydrofuran-CH₃OH—H₂O (3:1:1) was treated with LiOH H₂O(16 mg, 0.38 mmol, 2 equiv). The reaction mixture was stirred at 24° C.under N₂ for 6 h before the solvent was removed in vacuo. The residualsolid was mixed with H₂O (3 mL) and acidified with 1N aqueous HCl topH 1. The precipitate was collected by centrifugation and washed withH₂O (2×2 mL). Drying the solid in vacuo afforded 154 (47 mg, 47 mgtheoretical, 100%) as a pale yellow fluffy solid: mp >230° C. (dec); ¹HNMR (CF₃CO₂D, 400 MHz) 8.45 (d, 1H, J=9.2 Hz, C4-H), 7.83 (d, 1H, J=9.2Hz, C5-H), 4.40 (t, 1H, J=8.4 Hz, C2-H), 3.69 (t, 1H, J=8.4 Hz, C1-H₂);¹³C NMR (CF₃CO₂D, 100 MHz) 160.7 (C), 157.8 (C), 145.4 (C), 140.1 (C),130.6 (C), 130.2 (C), 121.7 (CH), 119.8 (C), 116.9 (CH), 50.8 (CH₂),26.9 (CH₂); IR (film)_(max) 3183, 1665, 1587, 1496, 1448, 1247, 1119cm⁻¹; FABHRMS (NBA) m/e 247.0838 (M⁺+H, C₁₁H₁₀N₄O₃ requires 247.0831).

Preparation of3-[(3′-Carbamoyl-1′,2′-dihydro-3′H-pyrrolo[3′,2′-e]benzoxazol-7′-yl)carbonyl]-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole(156) (Illustrated in FIG. 27). Phenol 14 (5.3 mg, 0.0159 mmol) wastreated with anhydrous 3M HCl-Ethylacetate (2 mL) at 24° C. for 30 min.The solvent was removed in vacuo to afford crude unstable 16(quantitative). A mixture of 16,[3-(dimethylamino)propyl]-ethylcarbodiimide hydrochloride(1-(3-DIMETHYLAMINOPROPYL)-3-ETHYLCARBODIIMIDE HYDROCHLORIDE (EDCI), 6.1mg, 0.032 mmol, 2 equiv), and CDPBO₁ 128 (3.7 mg, 0.015 mmol, 0.95equiv) was stirred in dimethylformamide (400 μL) at 24° C. under Ar for12 h. The solvent was removed in vacuo and the dry residue was mixedwith H₂O (1 mL) and stirred for 10 min. The precipitate was collected bycentrifugation, and washed with H₂O (2×1 mL) and dried in vacuo. Flashchromatography (SiO₂, 0.5×10 cm, 0-10% CH₃OH—CHCl₃ gradient elution)afforded 156 (6.4 mg, 7.3 mg theoretical, 88%) as a pale greenishpowder: mp >230° C. (dec); ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz) 10.57(s, 1H, OH), 8.21 (d, 1H, J=9.0 Hz, C4′-H), 8.14 (d, 1H, J=8.3 Hz,C6-H), 8.02 (s, 1H, C4-H), 7.86 (d, 1H, J=8.4 Hz, C9-H), 7.61 (d, 1H,J=9.0 Hz, C5′-H), 7.55 (t, 1H, J=7.7 Hz, C8-H), 7.40 (t, 1H, J=7.8 Hz,C7-H), 6.36 (br s, 2H, NH₂), 4.90 (d, 1H, J=10.5 Hz, C2-H), 4.78 (dd,1H, J=8.8, 11.9 Hz, C2-H), 4.23-4.25 (m, 1H, C1-H), 3.99-4.08 (m, 3H,CHHCl and C2′-H₂), 3.86 (dd, 1H, J=7.9, 10.9 Hz, CHHCl), 3.38-3.43 (m,2H, C1′-H₂); ¹³C NMR (DIMETHYLSULFOXIDE-d₆, 100 MHz) 156.1, 155.9,154.4, 154.1, 145.4, 142.9, 141.3, 136.7, 129.8, 127.6, 123.8, 123.2,123.1, 122.7, 121.8, 116.2, 114.5, 109.0, 100.0, 55.3, 48.2, 47.5, 41.2,25.3; IR (film)_(max) 3359, 3225, 1650, 1583, 1488, 1424, 1258, 1120,1024, 764 cm⁻¹; FABHRMS (NBA) m/e 463.1182 (M⁺+H, C₂₄H₁₉ClN₄O₄ requires463.1173).Natural (1S)-156: [α]³+44 (c 0.12, dimethylformamide).Ent-(1R)-156: [α]³−41 (c 0.09, dimethylformamide).

Preparation of3-[(3′-Carbamoyl-1′,2′-dihydro-3′H-pyrrolo[3′,2′-e]benzimidazol-7′-yl)carbonyl]-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole(158) (Illustrated in FIG. 27). Phenol 14 (4.6 mg, 0.0138 mmol) wastreated with anhydrous 3M HCl-Ethylacetate (2 mL) at 24° C. for 40 min.The solvent was removed in vacuo to afford crude unstable 16(quantitative). A mixture of 16,[3-(dimethylamino)propyl]-ethylcarbodiimide hydrochloride(1-(3-DIMETHYLAMINOPROPYL) 3-ETHYLCARBODIIMIDE HYDROCHLORIDE (EDCI), 5.3mg, 0.028 mmol, 2 equiv) and CDPBI₁ 154 (3.4 mg, 0.14 mmol, 1 equiv) wasstirred in dimethylformamide (400 μL) at 24° C. under N₂ for 6 h. Thesolvent was removed in vacuo. The dry residue was dissolved in 10%CH₃OH—CHCl₃ and loaded on a flash chromatography column (SiO₂, 0.8×10cm) and eluted with 0-10% CH₃OH—CHCl₃ gradient elution to afford 158(5.2 mg, 12.6 mg theoretical, 42%) as a light gray solid: mp >230° C.(dec); ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz) 13.16 (br s, 1H, NH),10.49 (br s, 1H, OH), 8.09-8.14 (m, 2H, ArH), 8.03 (d, 1H, J=9.0 Hz,ArH), 7.86 (d, 1H, J=8.3 Hz, C9-H), 7.52-7.56 (apparent t, 2H, J=8.7 Hz,ArH), 7.38 (t, 1H, J=7.4 Hz, C7-H), 6.25 and 6.21 (two s, 2H, NH₂), 5.17(d, 1H, J=10.4 Hz, C2-H), 4.83 (apparent t, 1H, J=9.2 Hz, C2-H), 4.24(m, 1H, C1-H), 4.02 (t, 3H, J=8.7 Hz, CHHCl, C2′-H, 3.83-3.88 (m, 1H,CHHCl), 3.27-3.37 (m, 2H, obscured by H₂O, Cl′-H₂); IR (film)_(max)3355, 3212, 2925, 1620, 1584, 1499, 1446, 1423, 1333, 1257, 1122, 1019cm⁻¹; FABHRMS (NBA) m/e 462.1345 (M⁺+H, C₂₄H₂₀ClN₅O₃ requires 462.1333)Natural (1S)-158: [α]³+49 (c 0.19, dimethylformamide).Ent-(1R)-158:[α]³−48 (c 0.04, dimethylformamide).

Preparation ofN²-[(3′-carbamoyl-1′,2′-dihydro-3′H-pyrrolo[3′,2′-e]benzimidazol-7′-yl)carbony]-1,2,9,9a-tetrahydro-cyclopropa-[c]-enz[e]-indol-4-one(162) or COMPOUND (160) (Illustrated in FIG. 27). A solution of 156 or158 (1.4 mg, 3 μmol) in 300 μL of tetrahydrofuran-dimethylformamide(1:1) was cooled to 0° C. and treated with DBN (0.5 μL, 4.5 μmol, 1.5equiv). The reaction mixture was slowly warmed to 24° C. and stirred for3.5 h. The mixture was placed on a flash chromatography column (SiO₂,0.5×3 mm), and eluted with 5-10% CH₃OH—CHCl₃ (gradient elution) toafford 160 or 162 (0.8 mg, 1.3 mg theoretical, 63%) as a bright yellowsolid. Selected representative data for 162: mp >230° C.; ¹H NMR(dimethylformamide-d₇, 400 MHz) 13.40 (br s, 1H, NH), 8.21 (d, 1H, J=8.9Hz, C4′-H), 8.10 (d, 1H, J=7.8 Hz, C5-H), 7.64 (t, 1H, J=7.5 Hz, C7-H),7.55 (d, 1H, J=8.9 Hz, C5′-H), 7.48 (t, 1H, J=8.0 Hz, C6-H), 7.31 (s,1H, C4-H), 7.29 (d, 1H, J=7.8 Hz, C8-H), 6.29 (br s, 2H, NH₂), 5.33 (d,1H, J=11.8 Hz, C1-H), 4.77 (dd, 1H, J=5.0, 11.8 Hz, C1-H), 4.20 (t, 1H,J=8.8 Hz, C2′-H₂), 3.44 (t, 2H, partially obscured by H₂O, J=8.8 Hz,C1′-H₂), 3.31-3.35 (m, 1H, C9a-H), 1.76-1.80 (m, 2H, C9-H₂); IR(film)_(max) 1656, 1620, 1589, 1495, 1442, 1406, 1272, 1125 cm⁻¹;FABHRMS (NBA) m/e 426.1545 (M⁺+H, C₂₄H₁₉N₅O₃ requires 426.1566). Natural(+)-162: [α]³+95 (c 0.04, dimethylformamide). Ent-(−)-162: [α]⁴−94 (c0.05, dimethylformamide).

DNA Alkylation Studies. General procedures, the preparation of singly³²P 5′ end-labeled double-stranded DNA, the agent binding studies, gelelectrophoresis, and autoradiography were conducted according toprocedures described in full detail elsewhere. (Boger et. al Tetrahedron1991, 47, 2661). Eppendorf tubes containing the 5′ end-labeled DNA (9μL, w794 and w836) in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) weretreated with the agent DIMETHYLSULFOXIDE (1 μL at the specifiedconcentration). The solution was mixed by vortexing and briefcentrifugation and susequently incubated at 25° C. for 3 days. Thecovalently modified DNA was separated from unbound agent by Ethanolprecipitation and resuspended in TE buffer (10 μL). The solution of DNAin an Eppendorf tube sealed with parafilm was heated at 100° C. for 30min to induce cleavage at the alkylation sites, allowed to cool to 25°C. and centrifuged. Formamide dye (0.33% xylene cyanol FF, 0.03%bromophenol blue, 8.7% Na₂EDTA 250 mM) was added (5 μL) to thesupernatant. Prior to electrophoresis, the sample was denatured bywarming at 100° C. for 5 min, placed in an ice bath, and centrifuged,before the supernatant (3 μL) was loaded directly onto the gel. Sangerdideoxynucleotide sequencing reactions were run as standards adjacent tothe reaction samples. Polyacrylamide gel electrophoresis (PAGE) was runon an 8% sequencing gel under denaturing conditions (8 M urea) in TBEbuffer (100 mM Tris, 100 mM boric acid, 0.2 mM Na₂EDTA) followed byautoradiography.

Preparation of compounds 164,166,168 (Illustrated in FIG. 29)Condensation of 3-nitrobenzaldehyde with methyl 2-azidoacetate (8 equiv,6 equiv NaOCH₃, CH₃OH, −23 to 0° C., 6 h, 88%) both reagentscommercially available from Aldrich, followed by thermolysis of theresulting methyl 2-azidocinnamate (xylene, reflux, 4.5 h, 81%) provideda readily separable mixture (4:1) of methyl 5- and7-nitroindole-2-carboxylate. For methyl 7-nitroindole-2-carboxylate(164): mp 122-125° C. (CH₂Cl₂, light yellow fine needles); ¹H NMR(CDCl₃, 400 MHz) 10.37 (br s, 1H, NH), 8.31 (d, 1H, J=8.0 Hz, C4-H),8.06 (d, 1H, J=8.0 Hz, C6-H), 7.36 (d, 1H, J=2.4 Hz, C3-H), 7.28 (t, 1H,J=8.0 Hz, C5-H), 3.99 (s, 3H, CO₂CH₃); IR (film)_(max) 3372, 1727, 1531,1445, 1344, 1298, 1249, 1188, 1107, 830, 763 cm⁻¹; FABHRMS (NBA) m/e221.0560 (M⁺+H, C₁₀H₈N₂O₄ requires 221.0562). For methyl5-nitroindole-2-carboxylate (168): ¹H NMR (DIMETHYLSULFOXIDE-d6, 400MHz) 12.65 (br s, 1H, NH), 8.73 (d, 1H, J=2.3 Hz, C4-H), 8.14 (dd, 1H,J=2.0, 8.0 Hz, C6-H), 7.60 (d, 1H, J=8.0 Hz, C7-H), 7.45 (d, 1H, J=0.7Hz, C3-H), 3.90 (s, 3H, CO₂CH₃); IR (film)_(max) 3316, 1701, 1614, 1531,1435, 1343, 1261, 1203, 992, 746 cm⁻¹. Similarly, condensation of4-nitrobenzaldehyde with methyl 2-azidoacetate (8 equiv, 6 equiv NaOCH₃,CH₃OH, −23 to 0° C., 7 h, 84%) followed by thermolysis (xylene, reflux,4 h, 83%) provided methyl 6-nitroindole-2-carboxylate (166): ¹H NMR(CDCl₃, 400 MHz) 9.27 (br s, 1H, NH), 8.39 (d, 1H, J=2.0 Hz, C7-H), 8.04(dd, 1H, J=2.0, 8.0 Hz, C5-H), 7.78 (d, 1H, J=8.0 Hz, C4-H), 7.28 (d,1H, J=2.3 Hz, C3-H), 4.00 (s, 3H, CO₂CH₃).

Preparation of compounds 170,172,174 (Illustrated in FIG. 29) Catalytichydrogenation of 164,166 or 168 (1 atm H₂, 0.1 wt equiv 10% Pd—C,Ethylacetate, 25° C., 4-5 h) provided the corresponding amines. Formethyl 7-aminoindole-2-carboxylate (170): 79%; mp 184° C. (dec, palegreen crystals); ¹H NMR (CDCl₃, 400 MHz) 9.47 (br s, 1H, NH), 7.21 (s,1H, C3-H), 7.20 (d, 1H, J=7.4 Hz, C6-H), 6.99 (t, 1H, J=7.5 Hz, C5-H),6.67 (d, 1H, J=7.4 Hz, C4-H), 3.97 (s, 3H, CO₂CH₃), 2.30 (br s, 2H,NH₂); IR (film)_(max) 3205, 2815, 1693, 1547, 1437, 1345, 1247, 1211,1112, 997, 827, 783, 734 cm⁻¹; FABHRMS (NBA) m/e 190.0747 (M⁺+H,C₁₀H₁₀N₂O₂ requires 190.0742). For methyl 6-aminoindole-2-carboxylate(172): 76%, ¹H NMR (CDCl₃, 400 MHz) 8.58 (br s, 1H, NH), 7.45 (d, 1H,J=8.4 Hz, C4-H), 7.11 (d, 1H, J=2.1 Hz, C3-H), 6.62 (d, 1H, J=1.9 Hz,C7-H), 6.59 (dd, 1H, J=1.9, 8.4 Hz, C5-H), 3.89 (s, 3H, CO₂CH₃), 3.79(br s, 2H, NH₂); IR (film)_(max) 3351, 2922, 1694, 1629, 1528, 1440,1271, 1206, 1130, 999, 834, 736, 668 cm⁻¹; FABHRMS (NBA) m/e 190.0740(M⁺+H, C₁₀H₁₀N₂O₂ requires 190.0742). For methyl5-aminoindole-2-carboxylate (174): 92%, mp 150-152° C. (CH₂Cl₂); ¹H NMR(CDCl₃, 400 MHz) 8.72 (br s, 1H, NH), 7.23 (d, 1H, J=8.6 Hz, C7-H), 7.03(dd, 1H, J=1.0, 2.1 Hz, C3-H), 6.93 (dd, 1H, J=1.0, 2.0 Hz, C4-H), 6.81(dd, 1H, J=2.0, 8.6 Hz, C6-H), 3.93 (s, 3H, CO₂CH₃), 3.57 (br s, 2H,NH₂); ¹³C NMR (CDCl₃, 100 MHz) 160.0 (C), 150.3 (C), 145.6 (C), 143.0(C), 127.7 (C), 117.7 (CH), 113.5 (CH), 112.6 (CH), 106.1 (CH), 52.2(CH₃); IR (film)_(max) 3320, 1691, 1628, 1531, 1437, 1376, 1337, 1232,1034, 997, 766 cm⁻¹; FABHRMS (NBA) m/e 190.0746 (M⁺+H, C₁₀H₁₀N₂O₂requires 190.0742).

General Method for the Preparation of Trimethylammonium SubstitutedIndole-2-carboxylate Methyl Esters: Methyl5-(Trimethylammonio)indole-2-carboxylate Iodides (176-180) (Illustratedin FIG. 29). Compound 174 (76 mg, 0.4 mmol) was dissolved indimethylformamide (3 mL) and treated with NaHCO₃ (168 mg, 2.0 mmol, 5equiv) and CH₃I (568 mg, 248 μL, 4.0 mmol, 10 equiv). The reactionmixture was stirred at 24° C. under N₂ for 4 h before the solvent wasremoved in vacuo. The dry residue was slurried in H₂O and precipitatewas collected by centrifugation. Recrystallization from CH₃CN afforded180 (129 mg, 144 mg theoretical, 90%) as a pale yellow solid: mp 228° C.(dec); ¹H NMR (DIMETHYLSULFOXIDE-d₆, 400 MHz) 12.37 (br s, 1H, NH), 8.26(d, 1H, J=2.6 Hz, C4-H), 7.89 (dd, 1H, J=2.6, 9.3 Hz, C6-H), 7.64 (d,1H, J=9.3 Hz, C7-H), 7.29 (s, 1H, C3-H), 3.86 (s, 3H, CO₂CH₃), 3.65 (s,9H, N(CH₃)₃); ¹³C NMR (DIMETHYLSULFOXIDE-d₆, 100 MHz) 161.4 (C), 140.6(C), 136.4 (C), 129.7 (C), 125.9(C), 116.9 (CH), 114.1 (CH), 113.9 (CH),108.9 (CH), 56.9 (three CH₃), 52.2 (CH₃); IR (film)_(max) 3446, 1708,1537, 1437, 1339, 1259, 1205, 995, 937, 770, 742 cm⁻¹; FABHRMS (NBA) m/e233.1290 (M⁺−I, C₁₃H₁₇IN₂O₂ requires 233.1290). Anal. Calcd forC₁₃H₁₇IN₂O₂: C, 43.35; H, 4.76; N, 7.78. Found: C, 42.99; H, 4.62; N,7.51.

Methyl 7-(trimethylammonio)indole-2-carboxylate Iodide (176) procedureas above except with 170: mp 151.5° C. (dec, pale green fine needles);¹H NMR (CD₃CN, 400 MHz) 10.27 (br s, 1H, NH), 8.07 (d, 1H, J=8.0 Hz,C4-H), 7.81 (d, 1H, J=8.0 Hz, C6-H), 7.53 (s, 1H, C3-H), 7.41 (t, 1H,J=8.0 Hz, C5-H), 4.05 (s, 3H, CO₂CH₃), 3.89 (s, 9H, N(CH₃)₃); ¹³C NMR(CD₃OD, 100 MHz) 162.8 (C), 133.5 (C), 133.2 (C), 131.5 (C), 128.0 (C),127.1 (CH), 121.7 (CH), 117.8 (CH), 111.2 (CH), 56.7 (three CH₃), 52.8(CH₃); IR (film)_(max) 3188, 1717, 1614, 1467, 1438, 1306, 1254, 1204,1149, 944, 833, 731 cm⁻¹; FABHRMS (NBA) m/e 233.1297 (M⁺−I, C₁₃H₁₇IN₂O₂requires 233.1290). Anal. Calcd for C₁₃H₁₇IN₂O₂: C, 43.35; H, 4.76; N,7.78. Found: C, 43.37; H, 4.73; N, 7.78.

Methyl 6-(Trimethylammonio)indole-2-carboxylate Iodide (178) procedureas above except with 172: mp 209° C. (dec, colorless crystals); ¹H NMR(DIMETHYLSULFOXIDE-d₆, 400 MHz) 12.48 (br s, 1H, NH), 7.91 (d, 1H, J=9.0Hz, C4-H), 7.80 (d, 1H, J=2.0 Hz, C7-H), 7.75 (dd, 1H, J=2.1, 9.1 Hz,C5-H), 7.25 (s, 1H, C3-H), 3.86 (s, 3H, CO₂CH₃), 3.68 (s, 9H, N(CH₃)₃);¹³C NMR (DIMETHYLSULFOXIDE-d₆, 100 MHz) 153.5 (C), 124.2 (C), 123.9 (C),122.2 (C), 118.7 (C), 117.8 (CH), 112.4 (CH), 108.5 (CH), 101.9 (CH),47.4 (three CH₃), 43.5 (CH₃); IR (film)_(max) 3409, 1716, 1605, 1564,1489, 1433, 1325, 1226, 1005, 942 cm⁻¹; FABHRMS (NBA) m/e 233.1290(M⁺−I, C₁₃H₁₇IN₂O₂ requires 233.1290). Anal. Calcd for C₁₃H₁₇IN₂O₂: C,43.35; H, 4.76; N, 7.78. Found: C, 43.36; H, 4.72; N, 7.81.

General Method for the Preparation of Trimethylammonium SubstitutedIndole-2-carboxylic Acids: 5-(Trimethylammonio)indole-2-carboxylic Acid(182,184,186) (Illustrated in FIG. 29). A solution of 180 (100 mg, 0.28mmol) in tetrahydrofuran-CH₃OH—H₂O (3:1:1, 2.6 mL) was treated withLiOH—H₂O (35 mg, 0.83 mmol, 3 equiv), and the reaction mixture wasstirred at 24° C. for 6 h. The solvent was removed and the dry residuewas mixed with H₂O (10 mL) and saturated aqueous NaCl (5 mL). Thesolution was acidified to pH 1 with the addition of 1N aqueous HCl andextracted with CH₃CN (10 mL each) until no UV active material wasdetected in aqueous solution. The extracts were combined, dried (Na₂SO₄)and concentrated. Recrystallization from CH₃CN afforded 186 (73.8 mg,96.2 mg theoretical, 77%) as a pale yellow solid: ¹H NMR(DIMETHYLSULFOXIDE-d₆, 400 MHz) 13.31 (br s, 1H, CO₂H), 12.19 (br s, 1,NH), 8.23 (d, 1H, J=1.8 Hz, C4-H), 7.88 (d, 1H, J=9.2 Hz, C6-H or C7-H),7.59 (d, 1H, J=9.2 Hz, C6-H or C7-H), 7.19 (d, 1H, J=1.1 Hz, C3-H), 3.65(s, 9H, N(CH₃)₃); ¹³C NMR (DIMETHYLSULFOXIDE-d₆, 100 MHz) 162.4 (C),140.5 (C), 136.3 (C), 131.3 (C), 126.1 (C), 116.5 (CH), 133.8 (two CH),108.2 (CH), 56.8 (three CH₃); IR (film)_(max) 3342, 3016, 1697, 1538,1469, 1419, 1339, 1226, 1194, 938, 852, 772 cm⁻¹; FABHRMS (NBA) m/e219.1143 (M⁺−Cl, C₁₂H₁₅ClN₂O₂ requires 219.1134.)

7-(Trimethylammonio)indole-2-carboxylic Acid (182) procedure as aboveexcept use 176: mp >198° C. (dec, white solid); ¹H NMR(DIMETHYLSULFOXIDE-d₆, 400 MHz) 13.41 (br s, 1H, CO₂H), 12.23 (br s, 1H,NH), 7.94 (d, 1H, J=7.9 Hz, C4-H), 7.74 (d, 1H, J=8.0 Hz, C6-H), 7.38(s, 1H, C3-H), 7.26 (t, 1H, J=8.0 Hz, C5-H), 3.79 (s, 9H, N(CH₃)₃); ¹³CNMR (DIMETHYLSULFOXIDE-d₆, 100 MHz) 162.2 (C), 132.2 (C), 131.5 (C),131.3 (C), 126.6 (C), 125.3 (CH), 120.1 (CH), 116.9 (CH), 109.5 (CH),55.5 (three CH₃); IR (film)_(max) 3327, 1694, 1477, I444, 1416, 1388,1328, 1173, 1141, 940, 730 cm⁻¹; FABHRMS (NBA) m/e 219.1141 (M⁺−Cl,C₁₂H₁₅ClN₂O₂ requires 219.1134).

6-(Trimethylammonio)indole-2-carboxylic Acid (184) procedure as aboveexcept use 178: mp >195° C. (dec, off-white needles); ¹H NMR(DIMETHYLSULFOXIDE-d₆, 400 MHz) 13.12 (br s, 1H, CO₂H), 12.33 (br s, 1H,NH), 7.88 (d, 1H, J=9.0 Hz, C4-H), 7.81 (d, 1H, J=2.2 Hz, C7-H), 7.73(d, 1H, J=2.2, 9.0 Hz, C5-H), 7.17 (d, 1H, J=1.7 Hz, C3-H), 3.66 (s, 9H,N(CH₃)₃); ¹³C NMR (DIMETHYLSULFOXIDE-d₆, 100 MHz) 162.3 (C), 143.7 (C),135.7 (C), 131.6 (C), 126.9 (C), 123.6 (CH), 112.5 (CH), 107.0 (CH),104.5 (CH), 56.5 (three CH₃); IR (film)_(max) 3260, 1689, 1530, 1328,1222, 1131, 835, 778 cm⁻¹; FABHRMS (NBA) m/e 219.1142 (M⁺−Cl,C₁₂H₁₅ClN₂O₂ requires 219.1134).

Preparation of3-[(Indol-2′-yl)carbonyl]-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole(seco-CBI-indole₁, 190) (Illustrated in FIG. 30). A sample of 14 (4.0mg, 0.012 mmol) was treated with anhydrous 4 M HCl-Ethylacetate (1 mL)at 25° C. for 30 min. The solvent was removed in vacuo to afford crudeunstable 16 (quantitative). A mixture of 16,[3-(dimethylamino)propyl]ethylcarbodiimide hydrochloride(1-(3-DIMETHYLAMINOPROPYL)-3-ETHYLCARBODIIMIDE HYDROCHLORIDE (EDCI), 5.8mg, 0.030 mmol, 2.5 equiv), and indole-2-carboxylic acid (188, 2.9 mg,0.018 mmol, 1.5 equiv) from Aldrich company in 0.2 mL ofdimethylformamide was stirred at 25° C. under Ar for 16 h. The mixturewas diluted with 0.3 mL of H₂O and extracted with Ethylacetate (0.4mL×4). The combined organic layer was concentrated. Chromatography(SiO₂, 40% Ethylacetate-hexane) afforded 190 (3.4 mg, 4.3 mgtheoretical, 79%) as a pale yellow solid: ¹H NMR (tetrahydrofuran-d₈,400 MHz) 11.04 (br s, 1H, NH), 9.31 (s, 1H, OH), 8.21 (d, 1H, J=8.3 Hz,C6-H), 8.02 (br s, 1H, C4-H), 7.78 (d, 1H, J=8.3 Hz, C9-H), 7.67 (d, 1H,J=7.9 Hz, C4′-H), 7.48 (dd, 1H, C8-H partially obscured by overlappingC7′-H), 7.47 (d, 1H, J=8.3 Hz, C7′-H), 7.30 (dd, 1H, J=8.0, 8.3 Hz,C7-H), 7.22 (dd, 1H, J=7.1, 8.3 Hz, C6′-H), 7.17 (s, 1H, C3′-H), 7.06(dd, 1H, J=7.1, 7.9 Hz, C5′-H), 4.78 (m, 2H, C2-H₂), 4.17 (m, 1H, C1-H),4.00 (dd, 1H, J=3.2, 11.1 Hz, CHHCl), 3.61 (m, 1H, CHHCl); IR(film)_(max) 3427, 3225, 3056, 2965, 2865, 1608, 1578, 1512, 1417, 1394,1363, 1338, 1316, 1252, 1140, 1058, 850, 804, 743 cm⁻¹; FABHRMS (NBA)m/e 377.1065 (M³⁰ +H, C₂₂H₁₇ClN₂O₂ requires 377.1057). Natural (1S)-2:[α]³+8.8 (c 0.17, tetrahydrofuran).

General Method for the Coupling of seco-N-BOC-CBI (14) with 15-17:3-[7′-((Trimethylammonio)indol-2′-yl)carbonyl]-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indoles(190,192,194,196) (Illustrated in FIG. 30). Phenol 14 (9.0 mg, 0.027mmol) was treated with anhydrous 3M HCl-Ethylacetate (2 mL) at 24° C.for 30 min. The solvent was removed in vacuo to afford crude unstable 16(quantitative). A mixture of 16,[3-(dimethylamino)propyl]ethylcarbodiimide hydrochloride(1-(3-DIMETHYLAMINOPROPYL)-3-ETHYLCARBODIIMIDE HYDROCHLORIDE (EDCI),10.3 mg, 0.054 mmol, 2.0 equiv), and 182 (9.3 mg, 0.027 mmol, 1.0 equiv)in 0.5 mL of dimethylformamide was stirred at 24° C. under Ar for 12 h.The solvent was removed in vacuo and the dry residue was mixed with H₂O(3 mL) and saturated aqueous NaCl (2 mL). The mixture was extracted withCH₃CN (5 mL×3). The organic layer was dried (Na₂SO₄) and concentrated.Chromatography (SiO₂, n-Butanol-H₂O-Ethylacetate-HOAc, 5:5:5:3) afforded192 (10.3 mg, 15.2 mg theoretical, 68%) as a pale yellow solid: mp >152°C. (dec); ¹H NMR (CD₃OD, 400 MHz) 8.22 (d, 1H, J=8.3 Hz, C6-H), 8.02 (d,1H, J=7.9 Hz, C4′-H), 7.97 (br s, 1H, C4-H), 7.81 (d, 1H, J=8.6 Hz,C6′-H or C9-H), 7.79 (d, 1H, J=8.4 Hz, C6′-H or C9-H), 7.55 (t, 1H,J=8.2 Hz, C8-H), 7.43 (s, 1H, C3′-H), 7.39 (t, 1H, J=8.2 Hz, C7-H), 7.35(t, 1H, J=8.0 Hz, C5′-H), 4.73-4.77 (m, 1H, C2-H), 4.65 (dd, J=1.7, 11.0Hz, C2-H), 4.17-4.21 (m, 1H, C1-H), 4.00 (dd, 1H, J=3.1, 11.2 Hz,CHHCl), 3.90 (s, 9H, N(CH₃)₃), 3.69 (apparent t, 1H, J=10.6 Hz, CHHCl);IR (film)_(max) 3354, 1624, 1584, 1466, 1414, 1326, 1259 cm⁻¹; FABHRMS(NBA) m/e 434.1648 (M⁺−Cl, C₂₅H₂₅Cl₂N₃O₂ requires 434.1635). Natural(1S)-3: [ ]³−9.9 (c 0.10, CH₃OH).

3-[6′-((Trimethylammonio)indol-2′-yl)carbonyl]-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole(194) procedure as above except use 186: ¹H NMR (CD₃OD, 400 MHz) 8.21(d, 1H, J=8.3 Hz, C6-H), 7.99 (d, 1H, J=8.8 Hz, C4′-H), 7.87 (br s, 1H,C4-H), 7.80 (d, 1H, J=8.3 Hz, C9-H), 7.65 (dd, 1H, J=2.3, 9.3 Hz,C5′-H), 7.64 (s, 1H, C7′-H), 7.54 (t, 1H, J=8.2 Hz, C8-H), 7.36-7.45 (m,1H, C7-H), 7.30 (s, 1H, C3′-H), 4.75-4.82 (m, 1H, C2-H), 4.70 (dd, 1H,J=1.8, 10.9 Hz, C2-H), 4.19-4.23 (m, 1H, C1-H), 4.00 (dd, 1H, J=3.2,11.2 Hz, CHHCl), 3.75 (s, 9H, N(CH₃)₃), 3.69 (dd, 1H, J=3.0, 11.2 Hz,CHHCl); IR (film)_(max) 3373, 1625, 1577, 1558, 1519, 1409, 1342, 1256cm⁻¹; FABHRMS (NBA) m/e 434.1722 (M⁺−Cl, C₂₅H₂₅Cl₂N₃O₂ requires434.1714). Natural (1S)-4: [ ]³+53 (c 0.04, CH₃OH).3-[5′-((Trimethlammonio)indol-2′-yl)carbonyl]-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole(196) procedure as above except use 188: ¹H NMR (CD₃OD, 400 MHz) 8.27(d, 1H, J=2.6 Hz, C4′-H), 8.21 (d, 1H, J=8.3 Hz, C6-H), 7.83 (br s, 1H,C4-H), 7.81 (dd, 1H, J=2.8, 9.3 Hz, C6′-H), 7.80 (d, 1H, J=8.3 Hz,C9-H), 7.73 (d, 1H, J=9.2 Hz, C7′-H), 7.53 (t, 1H, J=8.2 Hz, C8-H), 7.37(t, 1H, J=8.4 Hz, C7-H), 7.33 (s, 1H, C3′-H), 4.68-4.76 (m, 2H,partially obscured by H₂O, C2-H₂), 4.17-4.21 (m, 1H, C1-H), 3.99 (dd,1H, J=3.2, 11.2 Hz, CHHCl), 3.73 (s, 9H, N(CH₃)₃), 3.65 (dd, 1H, J=8.8,11.2 Hz, CHHCl); IR (film)_(max) 3374, 1557, 1416, 1342, 1265, 1232, 758cm⁻¹; FABHRMS (NBA) m/e 434.1619 (M⁺−Cl, C₂₅H₂₅Cl₂N₃O₂ requires434.1635). Natural (1S)-5: [ ]³+64 (c 0.10, CH₃OH).

DNA Alkylation Studies of 2-5: Selectivity and Efficiency. Eppendorftubes containing singly ³²P 5′-end-labeled double-stranded DNA¹⁰ (9 μL)in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) were treated with theagents 190-196 in DIMETHYLSULFOXIDE (1 μL, at the specifiedconcentrations). The solutions were mixed by vortexing and briefcentrifugation and subsequently incubated at 4° C. for 24 h. Thecovalently modified DNA was separated from unbound agent by Ethanolprecipitation of the DNA. The Ethanol precipitations were carried out byadding t-RNA as a carrier (1 μL, 10 μ/μL), a buffer solution containingsalt (0.1 volume, 3 M NaOAc in TE) and −20° C. Ethanol (2.5 volumes).The solutions were mixed and chilled at −78° C. in a REVCO freezer for 1h or longer. The DNA was reduced to a pellet by centrifugation at 4° C.for 15 min, washed with −20° C. 70% Ethanol (in TE containing 0.2 MNaCl) and recentrifuged briefly. The pellets were dried in a SavantSpeed Vac concentrator and resuspended in TE buffer (10 μL). Thesolutions of alkylated DNA were warmed at 100° C. for 30 min to inducecleavage at the adenine N3 alkylation sites. After brief centrifugation,formamide dye solution (5 μL) was added. Prior to electrophoresis, thesamples were denatured by warming at 100° C. for 5 min, placed in an icebath, centrifuged briefly, and the supernatant (2.8 μL) was loaded ontoa gel. Sanger dideoxynucleotide sequencing reactions were run asstandards adjacent to the agent treated DNA reaction samples.Polyacrylamide gel electrophoresis (PAGE) was run on an 8% sequencinggel under denaturing conditions (19:1 acrylamide:N,N′-methylenebisacrylamide, 8 M urea) in TBE buffer (100 mM Tris, 100mM boric acid, 0.2 mM Na₂EDTA). PAGE was pre-run for 30 min withformamide dye solution prior to loading the samples. Autoradiography ofdried gels was carried out at −78° C. using Kodak X-Omat AR film and aPicker Spectra™ intensifying screen.

1. A compound represented by the following structure:

wherein R₁ is selected from the group consisting of —CH₂CH₃ (alkyl),—NHCH₃ (—N-alkyl), —OCH₃ (O-alkyl), —NH₂, —NHNH₂, —NHNHCO₂ ^(t)Bu, and aradical represented by the following structure:

wherein: A is selected from the group consisting of NH and O; B isselected from the group consisting of C and N; R₂ is selected from thegroup consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl(C1-C6)₃ and a first N-substituted pyrrolidine ring; R₃ is selected fromthe group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl(C1-C6)₃, the first N-substituted pyrrolidine ring; R₄ is selected fromthe group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), and N-alkyl(C1-C6)₃; R₅ is selected from the group consisting of hydrogen,hydroxyl, O-alkyl (C1-C6), and N-alkyl (C1-C6)₃; and V₁ represents afirst vinylene group between R₂ and R₃; with the following provisos: ifR₂ participates in the first N-substituted pyrrolidine ring, then R₃also particlates in the first N-substituted pyrrolidine ring; if R₃participates in the first N-substituted pyrrolidine ring, then R₂ alsoparticlates in the first N-substituted pyrrolidine ring; if R₂ and R₃participate in the first N-substituted pyrrolidine ring, then R₄ and R₅are hydrogen; if R₂ is hydrogen, then R₄ and R₅ are hydrogen and R₃ isN-alkyl (C1-C6)₃; and wherein the first N-substituted pyrrolidine ringis fused to the first vinylene group between R₂ and R₃ and isrepresented by the following structure:

wherein: V₁ represents the first vinylene group between R₂ and R₃; R₆ isselected from the group consisting of —CH₂CH₃ (alkyl), —NHCH₃(—N-alkyl), —OCH₃ (O-alkyl), —NH₂, —NHNH₂, —NHNHCO₂ ^(t)Bu, and aradical represented by the following structure:

 wherein: C is selected from the group consisting of NE and O; D isselected from the group consisting of C and N; R₇ is selected from thegroup consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl(C1-C6)₃, and a second N-substituted pyrrolidine ring; R₈ is selectedfrom the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6),N-alkyl (C1-C6)₃, the second N-substituted pyrrolidine ring; V₂represents the second vinylene group between R₇ and R₈; with thefollowing provisos: if R₇ participates in the N-substituted pyrrolidinering, then R₈ also particlates in the N-substituted pyrrolidine ring; ifR₈ participates in the N-substituted pyrrolidine ring only if R₇ alsoparticlates in the N-substituted pyrrolidine ring; and wherein thesecond N-substituted pyrrolidine ring is fused to the second vinylenegroup between R₇ and R₈ and is represented by the following structure:

wherein: V₂ represents the second vinylene group between R₇ and R₈; R₉is selected from the group consisting of —CH₂CH₃ (alkyl), —NHCH₃(—N-alkyl), —OCH₃ (O-alkyl), —NH₂, —NHNH₂, and —NHNHCO₂ ^(t)Bu.
 2. Acompound represented by the following structure:

wherein X is selected from the group consisting of chlorine, bromine,iodine, and OTOS; and R₁ is selected from the group consisting of—CH₂CH₃ (alkyl), —NHCH₃ (—N-alkyl), —OCH₃ (O-alkyl), —NH₂, —NHNH₂,—NHNHCO₂ ^(t)Bu, and a radical represented by the following structure:

wherein: A is selected from the group consisting of NH and O; B isselected from the group consisting of C and N; R₂ is selected from thegroup consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl(C1-C6)₃ and a first N-substituted pyrrolidine ring; R₃ is selected fromthe group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl(C1-C6)₃, the first N-substituted pyrrolidine ring; R₄ is selected fromthe group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), and N-alkyl(C1-C6)₃; R₅ is selected from the group consisting of hydrogen,hydroxyl, O-alkyl (C1-C6), and N-alkyl (C1-C6)₃; and V₁ represents afirst vinylene group between R₂ and R₃; with the following provisos: ifR₂ participates in the first N-substituted pyrrolidine ring, then R₃also particlates in the first N-substituted pyrrolidine ring; if R₃participates in the first N-substituted pyrrolidine ring, then R₂ alsoparticlates in the first N-substituted pyrrolidine ring; if R₂ and R₃participate in the first N-substituted pyrrolidine ring, then R₄ and R₅are hydrogen; if R₂ is hydrogen, then R₄ and R₅ are hydrogen and R₃ isN-alkyl (C1-C6)₃; and wherein the first N-substituted pyrrolidine ringis fused to the first vinylene group between R₂ and R₃ and isrepresented by the following structure:

wherein: V₁ represents the first vinylene group between R₂ and R₃; R₆ isselected from the group consisting of —CH₂CH₃ (alkyl), —NHCH₃(—N-alkyl), —OCH₃ (O-alkyl), —NH₂, —NHNH₂, —NHNHCO₂ ^(t)Bu, and aradical represented by the following structure:

 wherein: C is selected from the group consisting of NH and O; D isselected from the group consisting of C and N; R₇ is selected from thegroup consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl(C1-C6)₃, and a second N-substituted pyrrolidine ring; R₈ is selectedfrom the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6),N-alkyl (C1-C6)₃, the second N-substituted pyrrolidine ring; V₂represents the second vinylene group between R₇ and R₈; with thefollowing provisos: if R₇ participates in the N-substituted pyrrolidinering, then R₈ also particlates in the N-substituted pyrrolidine ring; ifR₈ participates in the N-substituted pyrrolidine ring only if R₇ alsoparticlates in the N-substituted pyrrolidine ring; and wherein thesecond N-substituted pyrrolidine ring is fused to the second vinylenegroup between R₇ and R₈ and is represented by the following structure:

wherein: V₂ represents the second vinylene group between R₇ and R₈; R₉is selected from the group consisting of —CH₂CH₃ (alkyl), —NHCH₃(—N-alkyl), —OCH₃ (O-alkyl), —NH₂, —NHNH₂, and —NHNHCO₂ ^(t)Bu.
 3. Acompound represented by the following structure:

wherein A is selected from the group consisting of NH and O and B isselected from the group consisting of NH, O, and S.
 4. A compoundcompound represented by the following structure:


5. A compound compound represented by the following structures:


6. A compound compound represented by the following structure:


7. A compound compound represented by the following structure:


8. A compound compound represented by the following structure:

where R is selected from the group comprising of: H, 5-NMe₃ ⁺, 6-NMe₃ ⁺,7-NMe₃ ⁺.
 9. A compound compound represented by the following structure:

where R is selected from the group comprising of: CO₂ ^(t)Bu, H—HCl. 10.A compound compound represented by the following structure:


11. A compound compound represented by the following structure:


12. A compound compound represented by the following structure:


13. A compound compound represented by the following structure:


14. A compound compound represented by the following structure:

where R is selected from the group comprising of: H—HCl, CONHMe, CO₂CH₃,COEt.
 15. A compound compound represented by the following structure:


16. A compound compound represented by the following structure:

wherein A is selected from the group consisting of O and R is selectedfrom the group consisting of NO₂ and NH₂.
 17. A compound compoundrepresented by the following structure:

wherein B is selected from the group consisting of O and S.
 18. Acompound compound represented by the following structure:

wherein A is selected from the group consisting of NH and O and B isselected from the group consisting of NH, O, and S and R is selectedfrom the group consisting of H and CH₃.
 19. A compound compoundrepresented by the following structure:

wherein A is selected from the group consisting of NH and O and B isselected from the group consisting of NH, O, and S.
 20. A compoundcompound represented by the following structure:


21. A compound compound represented by the following structure:

where R is selected from the group comprising of: CO₂ ^(t)Bu, H—HCl. 22.A compound compound represented by the following structure:

where R is selected from the group comprising of: CO₂ ^(t)Bu, H.
 23. Acompound compound represented by the following structure:

where R is selected from the group comprising of: O^(t)Bu, NHNHCO₂^(t)Bu.
 24. A compound compound represented by the following structure: